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*
Department of Clinical Pharmacology, Jichi Medical School, Minamikawachi,
Tochigi, Japan.
Department of Pharmacology, Jichi Medical School, Minamikawachi, Tochigi,
Japan.
Department of Nephrology, Jichi Medical School, Minamikawachi, Tochigi,
Japan.
Correspondence to Dr. Shigeaki Muto, Department of Nephrology, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan. Phone: 81-285-58-7346; Fax: 81-285-44-4969; E-mail: smuto{at}jichi.ac.jp
Abstract
Abstract. To examine the functional significance of durg-transporting P-glycoprotein (P-gp), studies were conducted in the isolated and perfused proximal tubule S2 segment from mice disrupting both mdr1a and mdr1b genes [mdr1a/1b(—)(—)] and their wild-type mice. Efflux of the intracellular fluorescence of rhodamine 123, a fluorescence substrate of P-gp, into the lumen was measured, and the decay half-time of the intracellular fluorescence (T1/2) as an index of the drug-transporting P-gp activity was regarded. In the wild-type mice, the T1/2 was 34 ± 4 s (n = 36) at the basal period and was increased to 434 ± 41 s by the addition of luminal verapamil, a P-gp inhibitor. In the mdr1a/1b(—)(—) mice, the T1/2 was 407 ± 16 s (n = 10) at the basal period and was no longer affected by the luminal addition of verapamil. The digoxin content in the kidney after a repeated administration of the drug was markedly elevated in the mdr1a/1b(—)(—) mice. In conclusion, P-gp—mediated drug efflux capacity indeed exists in the apical membrane of proximal tubule cells from the wild-type mice, whereas it is absent in that of the mdr1a/1b(—)(—) mice.
P-glycoprotein (P-gp) was initially identified through its ability to confer multidrug resistance (MDR) in mammalian tumor cells (1). P-gp is a member of the ATP-binding cassette superfamily of transporters (1) and uses 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-gp, mdr1a (also called mdr3) and mdr1b (also called mdr1) (1,2,3). The mouse mdr1a gene is predominantly expressed in intestine, liver, and blood capillaries of brain and testis, whereas the mdr1b gene is predominantly expressed in adrenal glands, placenta, and ovaries (1). Similar levels of mdr1a and mdr1b expression are observed in the kidney (4,5). The mdr1a and mdr1b in the mouse together fulfill the same function as MDR1 in humans.
The expression of P-gp in the kidney is thought to occur exclusively in the apical membrane of the proximal tubule epithelium, where P-gp was expected to participate in the excretion of xenobiotics (6,7). Consistent with a role for P-gp as an excretory transporter, cell lines that express many properties of proximal tubule cells, including LLC-PK1 cells (8,9,10) and opossum kidney (OK) cells (11), primary cultures of flounder proximal tubule epithelial cells (12), cultured mouse proximal tubule cells (13), and intact killifish proximal tubules (12), exhibit net secretion of several MDR substrates, including vinblastine, verapamil, cyclosporin A, digoxin, and daunomycin. However, it is not known whether drug-transporting P-gp is functionally expressed in intact mammalian proximal tubule cells.
Drug metabolism in mammals is a complex process that involves parallel pathways that overlap with drug specificities. Although these can often be distinguished by the use of inhibitors, few inhibitors act on a single target. To resolve the problems, Schinkel et al. (3,5,14,15,16) recently generated mice with a genetic disruption of the genes, including the mdr1a and mdr1a/1b, and examined the physiologic and pharmacologic roles of these genes. However, no essential function of P-gp in the excretion of xenobiotics has been demonstrated in the kidney. The mice that lack the mdr1a gene [mdr1a(—) mice] have a normal lifespan when they are kept in the laboratory environment and are not challenged with P-gp—transported drugs (3). The mice that lack both mdr1a and mdr1b genes [mdr1a/1b(—)(—) mice] also have the same phenotype as their wild-type counterparts (16). However, the drug accumulation in the kidney of mdr1a(—) mice was identical to that of the wild-type mice (14,16), and mdr1b mRNA was expressed with a greater extent than that of their wild-type mice (5), whereas drug accumulation in the kidney of mdr1a/1b(—)(—) mice was greater than that of the wild-type mice (3). These findings suggest the possibility that the mdr1b gene but not the mdr1a gene may be important for P-gp—mediated drug excretion in the kidney. However, there is no direct evidence of this possibility. Therefore, the present study used mdr1a/1b(—)(—) mice. In combination with in vitro microperfusion technique and measurements of intracellular fluorescence of rhodamine 123 (R-123), a fluorescence substrate of P-gp, the present study was therefore designed to examine whether drug-transporting P-gp is functionally expressed in the isolated perfused proximal tubule S2 segment from the wild-type and mdr1a/1b(—)(—) mice.
Materials and Methods
Animals
The mdr1a/1b(—)(—) mice used in this study were originally
described by Schinkel et al.
(3). Male
mdr1a/1b(—)(—) mice and FVB (wild-type) mice served as background
(body weight, 25 to 40 g) and were purchased from Taconic Engineering
(Germantown, NY). These 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 knockout animals were matched
with their wild-type controls.
In Vitro Microperfusion
The mice from both groups were anesthetized with intraperitoneal injection
of pentobarbital sodium (4 mg/100 g body wt), and both kidneys were removed.
Slices of 1 to 2 mm were taken from the coronal section of each kidney and
transferred to a chilled dish containing modified Collins' solution having the
following composition: 14 mM KH2PO4, 14 mM
K2HPO4, 15 mM KCl, 9 mM NaHCO3, and 160 mM
sucrose (pH 7.4). 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). Each tubule was
perfused in vitro according to the method of Burg et al.
(17) with slight modifications
in our laboratory
(18,19,20).
The details of the technique have been published previously
(18,19,20);
accordingly, they will be presented here only briefly. After suspending the
tubules between the two pipettes, the lumen was perfused at 10 to 20 nl/min by
changing the height of the reservoir connected to the back end of the
perfusion pipette. A system of the flowing-through bath was used to permit
rapid exchange of the bathing fluid. The flow rate of the bathing fluid was in
a range of 5 to 10 ml/min. A water jacket was used to maintain the bath
temperature at 37°C. The composition of the control luminal and bathing
solution used in the study was as follows: 110 mM NaCl, 5 mM KCl, 25 mM
NaHCO3, 0.8 mM Na2HPO4, 0.2 mM
NaH2PO4, 10 mM Na-acetate, 1.8 mM CaCl2, 1.0
mM MgCl2, 8.3 mM glucose, and 5 mM alanine. The control solution
had an osmolality between 285 and 295 mOsm/kg H2O and was
equilibrated with 95% O2/5% CO2 and adjusted to pH 7.4
at 37°C.
Measurement of Efflux of P-gp Substrate
P-gp—mediated drug transport was assessed by measuring the efflux of
R-123,
(13,21).
After the tubules were hooked up to the perfusion system, they were perfused
with the control solution. After a 30-min equilibration period, the tubules
were perfused with the control solution containing 10 µM R-123 at 37°C
for 15 to 20 min, and the measurement of intracellular R-123 fluorescence was
started. Luminal perfusate was then rapidly changed to the control solution
without R-123. In the preliminary study, no reduction in the intracellular
R-123 fluorescence signal was detected until R-123 was removed from the lumen.
Intracellular fluorescence signal of the dye from a focused single cell was
monitored by microscopic fluorescence photometry system (OSP-3, Olympus,
Tokyo, Japan) 100 times per minute. Perfused tubules were positioned at the
bottom of the bath chamber as closely as possible and were viewed with a
40x fluorescence objective. The diameter of the beam of light focused on
the single tubule cell was 8.5 µm. The light source was a 75 W xenon lamp
(18). The R-123 fluorescence
was excited at 490 nm, and fluorescence emission was measured at 530 nm. To
minimize dye bleaching and cell damage, protocols were made as short as
possible. The decay of the intracellular R-123 fluorescence upon rapid removal
of the dye was followed as described previously
(13,21).
Because there is R-123 mitochondrial accumulation and quenching at high
concentrations, decay of the dye was determined at the linear range of the
fluorescence-concentration relationship, approximately 50% of the maximal
intracellular fluorescence
(13,21).
The apparent rate constants for R-123 decay (k) were obtained by
using PH/EDISM® (Mediware, Amsterdam, the Netherlands), a computer program
originating from pharmacokinetic analysis, to fit the data to the following
equation
(13,21)
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Determination of Digoxin Concentration in Serum and Kidney from
Wild-Type and mdr1a/1b(—)(—) Mice
For this purpose, the mice from both groups at 14 to 20 wk old were used. A
2.0-mg/ml stock solution of digoxin was prepared with a 0.1-M glucose solution
containing 2.6 M propylene glycol and 8.7 M ethanol. It was then diluted with
a 0.3-M glucose solution to make a final concentration of 0.05 mg/ml for
intraperitoneal injection. Digoxin (20 µg/100 g body wt) was
intraperitoneally injected once a day into the mice for 7 d. Twenty-four h
after the final dosing, the animals were anesthetized with intraperitoneal
pentobarbital sodium (5 mg/100 g body wt). Blood samples were then collected
from the inferior vena cava. Both kidneys were removed after blood had been
flushed out with saline and were weighed and homogenized with distilled water
for assay of digoxin content. The digoxin concentrations in serum and tissue
homogenate were measured by an automatic fluorescence polarization immunoassay
(TDx autoanalyzer, Dinabot Co., Ltd., Tokyo, Japan)
(22).
Chemicals
R-123 was purchased from Molecular Probe (Eugene, OR). Others were obtained
from Sigma (St. Louis, MO) with highest grade. Verapamil was dissolved in DMSO
at 1 mg/ml final concentration. Tetraethylammonium (TEA) was dissolved in
distilled water at 1 mg/ml final concentration. Equivalent concentrations of
vehicle were added as a control for individual protocols.
Statistical Analyses
All of the data are expressed as the mean ± SEM. Comparisons were
performed by t test, one-way ANOVA in combination with Fisher's
protected least significant difference, and Mann-Whitney U test, as
needed, P < 0.05 was regarded as significant.
Results
Efflux of the Intracellular R-123 Fluorescence in the Proximal Tubule
S2 Segment from the Wild-Type Mice
First, we measured intracellular fluorescence signal of R-123 as a function
of time in proximal tubule cells from the wild-type mice. A representative
record of its intracellular fluorescence is shown in
Figure 1A. Results from the
efflux experiments are summarized in Figure
2. There was no change in intracellular fluorescence signal before
the dye was washed out from the lumen. However, upon rapidly removing the dye
from the lumen, the decay of the intracellular fluorescence was observed and
followed a single exponential function of time, as described previously
(13,21).
The calculated T1/2 was 34 ± 4 s (n = 36), a value
compatible with that previously reported in mouse proximal tubule cell line
expressing P-gp (13). The
T1/2 was significantly (P < 0.001) prolonged to 434
± 41 s (n = 11) by the addition of luminal verapamil (5
µM), an inhibitor of P-gp, and was completely reversible after removal of
the drug. Also, the apparent rate constants were significantly (P
< 0.001) decreased from 1.22 ± 0.25 to 0.09 ± 0.03
min-1 (n = 11) after the addition of verapamil.
Conversely, the T1/2 was not altered at all by the addition of
luminal TEA (1 mM), a model substrate for renal organic cation transport
system (Figures 1B and
2). The longer exposure to TEA
also caused no effect on T1/2 (data not shown). These findings
indicate that R-123 is indeed extruded from the cell into the lumen via
P-gp.
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Efflux of the Intracellular R-123 Fluorescence in the Proximal Tubule
S2 Segment from the mdr1a/1b(—)(—) Mice
Next, we examined whether the P-gp—mediated drug efflux is present in
the proximal tubule cell from mdr1a/1b(—)(—) mice. A
representative record of intracellular R-123 fluorescence is shown in
Figure 3A. Results from the
efflux experiments are summarized in Figure
3B. The T1/2 at the basal period was 407 ± 16 s
(n = 10), a value significantly (P < 0.001) greater than
that of the wild-type mice. The apparent rate constants (0.11 ± 0.03
min-1, n = 10) were significantly (P < 0.001)
smaller than those of the wild-type mice (1.22 ± 0.25 min-1,
n = 11). In sharp contrast to the wild-type mice, the T1/2
was no longer affected by the addition of luminal verapamil (5 µM;
Figure 3). Also, in the
knockout mice, the apparent rate constants (from 0.11 ± 0.03 to 0.09
± 0.03 min-1, n = 10) were unchanged after the
addition of verapamil. The values of T1/2 and the apparent rate
constants seen after the addition of verapamil in the wild-type mice were
similar to those of the knockout mice, indicating that the
mdr1a/1b(—)(—) mice never possess drug-transporting P-gp activity
at the apical membrane of proximal tubule cells.
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Digoxin Content in the Kidney from Knockout and Their Wild-Type
Mice
Finally, we examined whether the digoxin content in the kidney from
mdr1a/1b(—)(—) mice is actually affected, because digoxin, a model
of P-gp substrate drug, is excreted from kidneys. Experiments were performed
in five wild-type and mdr1a/1b(—)(—) mice. Average body and kidney
weights, respectively, were 28.0 ± 0.7 g and 424 ± 31 mg in
wild-type mice and 26.2 ± 0.8 g and 439 ± 29 mg in
mdr1a/1b(—)(—) mice, values not significantly different between
the genotypes. As shown in Figure
4, the kidney digoxin content was significantly higher in the
mdr1a/1b(—)(—) mice (12.3 ± 7.4 pg/mg kidney, n =
5, P < 0.001) than in their wild-type mice (0.4 ± 0.3 pg/mg
kidney, n = 5). These findings confirm a previous report
(3). Conversely, serum digoxin
concentrations in the knockout mice (4.52 ± 0.76 ng/ml, n = 5,
P = 0.136) were slightly higher but not significantly different from
those of the wild-type mice (2.15 ± 1.21 ng/ml, n = 5).
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Discussion
In combination with in vitro microperfusion techniques and measurements of intracellular R-123 fluorescence, the present study examined whether drug-transporting P-gp is actually expressed in the apical membrane of proximal tubule cells from wild-type and mdr1a/1b(—)(—) mice. As shown in Figure 1, upon rapid removal of the dye from the lumen, the decay of the intracellular R-123 fluorescence was observed and followed a single exponential function of time. Conversely, the intracellular R-123 fluorescence was unchanged before the dye was washed out from the lumen. Also, when the dye was rapidly removed from the lumen, the decay of the intracellular fluorescence was observed in the wild-type mice (see Figure 1), whereas it was not found in the knockout mice (see Figure 3). Therefore, the decay of the intracellular R-123 fluorescence is not due to photobleaching but due to the drug-transporting P-gp activity. Our data demonstrate that the P-gp—mediated drug efflux mechanism indeed exists in the apical membrane of proximal tubule cells from the wild-type mice but lacks in that of the mdr1a/1b(—)(—) mice. This is the first study to demonstrate that disruption of both mdr1a and mdr1b genes causes lack of drug-transporting P-gp activity in the proximal tubule cell.
In mdr1a(—) mice, the cumulative urinary excretion of the P-gp substrate digoxin was increased compared with their wild-type mice (14). This could be due to the activity of other transporters, e.g., mdr1b P-gp, for which the mRNA levels were increased in the mice with disrupted mdr1a gene (5). In sharp contrast to the mdr1a(—) mice, in the mdr1a/1b(—)(—) mice, the kidney digoxin content was significantly increased compared with that of the wild-type mice (see Figure 4) (3). In the proximal tubule from the mdr1a/1b(—)(—) mice, the decay half-time of intracellular R-123 was markedly increased and the apparent rate constants were markedly decreased, and the increased decay half-time or the decreased apparent rate constants were no longer affected by the addition of luminal verapamil. These results indicate that in the proximal tubule from the mdr1a/1b(—)(—) mice, the P-gp—mediated drug efflux capacity is completely lost and other transporters do not compensate for the absence of mdr1a/1b P-gp. Taken together with our findings and other reports, the mdr1b gene but not the mdr1a gene could play an important role in the maintenance of drug-transporting P-gp activity in the proximal tubule.
In conclusion, we clearly demonstrated that drug-transporting P-gp activity is actually expressed in the apical membrane of the proximal tubule from the wild-type mice, whereas it abolishes in that of the mdr1a/1b(—)(—) mice. These findings will be helpful to get a better understanding of the physiologic role of P-gp on the xenobiotic efflux in normal kidney epithelial cells.
Acknowledgments
We appreciate Ms. Mariko Hojo for her excellent technical assistance.
This work was supported by a grant from the Japanese Kidney Foundation (Jinkenkyukai) and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports, Japan.
A portion of this work was presented at the Annual Meeting of the American Society of Nephrology in Miami Beach and has been published in abstract form
References
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