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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 282:1518 –1525, 1997
Vol. 282, No. 3
Printed in U.S.A.
Disposition of Morphine in the Rat Isolated Perfused Kidney:
Concentration Ranging Studies1
KATHRYN M. SHANAHAN, ALLAN M. EVANS and ROGER L. NATION
Centre for Pharmaceutical Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia
Accepted for publication May 6, 1997
In humans and experimental animals, morphine is extensively metabolized via conjugative and oxidative pathways to
metabolites that exhibit pharmacological activity (Milne et
al., 1996). Although only 10% of a dose of morphine is excreted unchanged in urine, the kidney plays a major role in
the excretion of M3G, M6G (Milne et al., 1992; Somogyi et al.,
1993) and possibly normorphine (Milne et al., 1996).
The renal clearance of morphine, which exists predominantly as a cation at physiological pH, involves filtration at
the glomerulus, tubular secretion and possibly reabsorption.
A significant degree of tubular secretion has been observed in
humans (Milne et al., 1992; Somogyi et al., 1993), chickens
(May et al., 1967; Watrous et al., 1970; Hakim and Fujimoto,
1971) and sheep (Milne et al., 1995). These findings are
supported by in vitro studies involving the stop-flow microperfusion of the renal proximal tubule from the rat (Ullrich and Rumrich, 1995), cortical slices from mice (Teller et
Received for publication November 18, 1996.
1
This study was supported by the National Health and Medical Research
Council of Australia, grant number 940330.
morphine within the kidney was high (31 6 3 ml/g at 0.2 mM),
which indicates extensive accumulation, and remained constant with increasing perfusate concentration. The ratio of unbound renal excretory clearance to glomerular filtration rate
was always greater than unity for all kidneys, which indicates
that the renal excretion of morphine involves net tubular secretion. This ratio was constant (P . .05) over the 100-fold concentration range of the single-dose study. In the multiple-dose
study, the ratio was marginally but significantly (P , .05) higher
at concentrations of 2, 20 and 200 mM than at 0.2 mM, a
difference that cannot be explained by saturation of tubular
secretion. The results suggest that the tubular secretion of
morphine is not saturated over a wide range of concentrations
(0.2–200 mM).
al., 1976) and proximal tubular segments from the rabbit
(Schäli and Roch-Ramel, 1982). Furthermore, secretion has
been observed in the IPK of the rat (Ratcliffe et al., 1985; van
Crugten et al., 1991). In the chicken (May et al., 1967;
Watrous et al., 1970) and rabbit (Schäli and Roch-Ramel,
1982), secretion of morphine involves a cation transport system, as evidenced by competition between morphine and
other organic cations, such as cyanine 863 (May et al., 1967;
Watrous et al., 1970), mepiperphenidol (Watrous et al., 1970;
Schäli and Roch-Ramel, 1982) and quinine (Schäli and RochRamel, 1982). Studies in humans (Somogyi et al., 1993) and
the rat IPK (van Crugten et al., 1991) suggest that the renal
clearance of morphine also involves a component of reabsorption, and Ullrich and Rumrich (1995) recently suggested that
the renal disposition of morphine involves carrier-mediated
bidirectional tubular transport.
Drug metabolizing enzymes, such as UDP-glucuronosyltransferase, sulfotransferase and cytochromes P-450, are
known to exist in the mammalian kidney (Jones et al., 1979;
Anders, 1980; Hjelle et al., 1986), and morphine is one of
90
ABBREVIATIONS: Ae90
0 , total amount of morphine excreted unchanged in urine between 0 and 90 min; AUC0 , area under the perfusate morphine
`
concentration vs. time curve from 0 to 90 min; AUC0 , area under the perfusate morphine concentration vs. time curve from time zero to infinity;
BSA, bovine serum albumin; CLM, total organ clearance of morphine; CLM
R , renal excretory clearance of morphine in a urine collection interval;
M
CLR(0
–90), renal excretory clearance of morphine from 0 to 90 min; fu, fraction of morphine unbound in perfusate; IPK, isolated perfused kidney;
M3G, morphine-3-glucuronide; M6G, morphine-6-glucuronide; %TR, percent tubular reabsorption; T1/2, half-life; UFR, urine flow rate; VM
K , volume
of distribution of morphine within the kidney; Vp, perfusate volume.
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ABSTRACT
The rat isolated perfused kidney was used to investigate the
linearity of the renal disposition of morphine and its potential
oxidative and glucuronidative metabolism by the kidney. In a
set of single-dose experiments, morphine was administered to
recirculating perfusion medium to achieve initial concentrations
of 0.2, 2 and 20 mM (n 5 4 at each concentration). In a set of
multiple-dose experiments, morphine was administered to perfusate as sequential bolus doses to achieve concentrations of
0.2, 2, 20 and 200 mM (n 5 6). HPLC was used to determine the
concentration of morphine in perfusate and urine. Normorphine, morphine-3-glucuronide and morphine-6-glucuronide
could not be detected in perfusate or urine, a result that suggests an absence of oxidative and glucuronidative metabolism
of morphine by the rat kidney. The volume of distribution of
1997
Renal Disposition of Morphine
Materials and Methods
Chemicals
The following drugs and chemicals were used in this study: morphine hydrochloride (MacFarlane Smith Ltd., Edinburgh, UK), normorphine hydrochloride (Makor Chemicals, Jerusalem, Israel),
[14C]-carboxy-inulin (3 mCi/g; Du Pont, Dreiech, West Germany),
fraction V BSA (Miles Diagnostics Inc., Kankakee, IL), L-cysteine,
glycine, L-glutamic acid and D-mannitol (Sigma Chemical Co. (St.
Louis, MO), D-glucose and n-butyl alcohol of HPLC grade (E. Merck,
Darmstadt, Germany) and acetonitrile of “far UV” grade and methanol and chloroform of HPLC grade (BDH Laboratory Supplies,
Poole, England). All other chemicals were of analytical grade or
equivalent.
Rat IPK
The study was approved by the Institute of Medical and Veterinary Science Animal Ethics Committee (Adelaide, South Australia).
Male Sprague-Dawley rats (345–505 g) from the Gilles Plains Animal Resource Centre (Adelaide, Australia) were maintained at approximately 21°C on a 12-hr light/dark cycle, with free access to food
and water. The IPK preparation was based on methods described
previously (Ellis et al., 1990; Mancinelli et al., 1995). Rats were
anesthetised with an i.p. injection of sodium pentobarbital (60 mg/kg
Nembutal; Boehringer Ingelheim, Artarmon, NSW, Australia) before
surgery. The abdominal cavity was opened to expose the right renal
artery, and the surgical procedure of Mancinelli et al., (1995) was
used thereafter.
The perfusate medium (150 ml) consisted of erythrocyte-free
Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 65 g/l of BSA,
D-glucose (5 mM), L-cysteine (0.5 mM), glycine (2.3 mM) and L-
glutamic acid (0.5 mM). Before use, BSA was dissolved in KrebsHenseleit buffer (130 g/l) and purified by dialyzing against three
exchanges of protein-free buffer over 3 days at 4°C. Dialyzed albumin
solutions were stored frozen at 220°C. On the day of a perfusion
experiment, the concentration of BSA in the perfusion medium was
adjusted to 65 g/l, and the three amino acids and glucose were added.
Perfusate was then filtered successively through 1.2-mm and
0.45-mm filters (Millipore, Bedford, MA). Perfusate was recirculated
through the perfusion system, before cannulation of the kidney, for
at least 30 min, which enabled the perfusate to become adequately
oxygenated (.0.6 mM). [14C]-inulin (0.35 mCi) was added to the
perfusate reservoir 15 to 20 min after the kidney was cannulated,
and morphine was then added to the perfusate in accordance with
the protocols described below. Urine was collected into preweighed
tubes, and the volume was determined gravimetrically. Perfusate
was collected from the reservoir at the midpoint of each urine collection interval or as specified. Urine and perfusate samples were
stored at 220°C until analysis. Upon completion of a perfusion
experiment, the kidney was weighed.
Perfusate was delivered to the kidney via a rotary pump (Masterflex model 7521-35, Cole Palmer, Chicago, IL), an 8-mm in-line filter
(Millipore), a silastic tubing oxygenator, a glass bubble-trap, a flow
meter and finally a glass cannula. Venous outflow drained directly
into the perfusate reservoir. Renal arterial pressure was maintained
at 100 6 15 mm Hg by adjustment of the perfusate flow rate (35–50
ml/min). The concentration of oxygen in perfusate flowing into the
kidney was determined routinely, using an Orion Model 820 Dissolved Oxygen Meter (Boston, MA), to be in excess of 0.6 mM.
Functional viability and performance of the kidney were assessed
by measurement of the GFR, determined as the renal clearance of
[14C]-inulin), UFR, perfusion pressure, perfusate pH, urine pH and
the %TR of water, glucose and sodium.
Experimental Design
A: Single-dose IPK studies. Approximately 5 min after the
addition of [14C]-inulin, morphine was administered as a bolus dose
into the perfusate reservoir, to achieve an initial concentration of 0.2
(low, n 5 4), 2 (medium, n 5 4) or 20 mM (high, n 5 4), respectively.
Urine samples were collected over 10-min intervals for 90 min, and
perfusate samples (1.4 ml) were collected immediately after dosing
and thereafter at 0.5, 1, 2.5 and 5 min and at the midpoint of each
urine collection interval. We determined fu at concentrations of 0.1,
1 and 10 mM at 37°C by ultrafiltration of four replicates at each
concentration through YMT membranes using the MPS-1 Micropartition system (Amicon Corp, Danvers, MA).
B: Multiple-dose IPK studies. Multiple-dose IPK studies (n 5 6)
consisted of four periods (0–15, 15–30, 30–45 and 45–60 min) in
which morphine was administered to the perfusate reservoir as a
series of bolus doses to achieve initial morphine concentrations of 0.2
mM (low), 2 mM (medium), 20 mM (high) and 200 mM (extra-high),
respectively. Before the addition of the initial morphine bolus dose,
[14C]-inulin was added to the perfusate reservoir. For the first 5 min
after each bolus dose, morphine was allowed to equilibrate. To make
possible calculation of CLM
R after each bolus dose, urine was collected
over the final 10 min of each of the four periods, and perfusate was
sampled every 5 min. The binding of morphine to perfusate protein
was studied at concentrations of 0.2, 2, 20 and 200 mM, as described
above.
Analytical Methods
The concentration of morphine in perfusate was determined using
a validated reverse-phase HPLC method that utilized normorphine
as the internal standard. Before analysis, perfusate samples (0.5 ml)
collected during the medium, high and extra-high periods were diluted 1 in 2, 1 in 20 and 1 in 200, respectively, in blank perfusate.
Each sample was supplemented with internal standard (200 ml of 1
mg/ml normorphine) and 0.2 M bicarbonate buffer pH 9 (0.5 ml).
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many drugs that has been suggested to undergo renal metabolism. Indeed, evidence exists for the glucuronidation of
morphine by microsomes prepared from fetal (Pacifici and
Rane, 1982) and adult (Yue et al., 1988; Cappiello et al., 1991)
human kidneys, and by proximal tubules isolated from the
rabbit (Schäli and Roch-Ramel, 1982). In addition, studies
conducted in vivo in sheep (Milne et al., 1993; Milne et al.,
1995) and dogs (Jacqz et al., 1986) have provided evidence for
glucuronidation of morphine by the kidney. In contrast, glucuronidation of morphine was not observed in rat kidney
microsomes (Rush et al., 1983; Rush and Hook, 1984) or the
rat IPK (Ratcliffe et al., 1985; van Crugten et al., 1991).
However, after studying the disposition of morphine in intact
rats and rats in which the bile duct was cannulated and renal
pedicles were ligated, Horton and Pollack (1991) hypothesized that the kidney is involved in the metabolic clearance of
morphine. Hence some controversy remains about the role of
the rat kidney in the metabolism of morphine. It is possible
that the apparent conflict between the studies conducted in
rats is due to the formation by the kidney of other metabolites, such as normorphine.
To date, the effect of alterations in concentration on morphine renal transport has not been investigated. In view of
the significant degree of renal tubular secretion of morphine,
it is conceivable that the relative contribution of the three
renal clearance mechanisms (i.e., filtration, secretion and
reabsorption) is concentration-dependent. Hence the present
studies were designed to investigate the effect of concentration of morphine on its renal disposition in the rat IPK. An
additional aim was to resolve the uncertainty surrounding
the role of the rat kidney in the metabolism of morphine via
conjugative and oxidative routes.
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Shanahan et al.
Vol. 282
Data Analysis
For the single-dose and multiple-dose studies, the CLM
R was determined as the rate of excretion into urine divided by the midpoint
perfusate concentration, calculated according to equation 1.
CLRM 5
concentration of morphine in urine 3 UFR
concentration of morphine in perfusate
(1)
In single-dose studies, CLM
R was calculated for the eight urine collection intervals from 10 to 90 min after the administration of morphine, whereas in the multiple-dose studies, CLM
R was calculated
over the final 10 min after each bolus dose, for the four urine
collection intervals. The renal clearances of glucose, sodium and
[14C]-inulin were calculated in an analogous manner, the latter providing an estimate of GFR. The %TR of water, glucose and sodium
was calculated according to equation 2.
S
%TR 5 1 2
D
X
z 100
GFR
(2)
where X is either UFR for the calculation of %TR of water or renal
clearance for the determination of %TR of glucose or sodium. We
calculated fu from the ultrafiltration experiments according to equation 3.
fu 5
concentration of morphine in ultrafiltrate of perfusate
concentration of morphine in unfiltered perfusate
(3)
Indices of the net tubular transport of morphine were obtained from
M
the ratio of CLM
R to the product of fu and GFR (CLR /fu z GFR) and the
difference between total excretory morphine clearance and the component of this clearance that arose from glomerular filtration (CLM
R 2
fu z GFR).
M
For the single-dose studies, the time-averaged CLR(0
–90) was calculated according to equation 4.
CLRM~0 –90! 5
Ae090
AUC090
(4)
CLM in the single-dose studies was calculated according to equation
5.
CLM 5
dose
AUC0`
(5)
where AU`
0 is calculated using the trapezoidal rule for the observed
values and then extrapolated to infinity. The volume of distribution
of morphine within the kidney (VM
K ) was calculated according to
equation 6.
VM
K 5
S
D
T1/2 3 CLM
2 VP
0.693
(6)
where T1/2 was determined from the log-linear terminal portion of
the perfusate concentration-time curve, and VP is the actual volume
of perfusion medium within the reservoir and the perfusion circuit.
Data are presented as mean 6 S.D. Analysis of variance was used
to test for differences that were considered significant at the level of
.05. Factorial design ANOVA was used to assess the concentrationdependence of protein binding for the single- and multiple-dose studies and to assess whether there were significant differences between
the three groups of single-dose IPKs in terms of kidney function and
the pharmacokinetic parameters for morphine. In addition, repeated-measures ANOVA was performed to determine whether there
were any significant changes in these parameters with time. For the
multiple-dose IPK studies, repeated-measures ANOVA was used to
test for changes in kidney function parameters and morphine renal
clearance measurements, across the four perfusion periods. Upon
detection of a significant difference, pairwise comparisons were performed using the Fisher test. Simple bivariate regression analyses
were performed to determine the relationship between morphine
renal clearance parameters and functional parameters such as UFR,
GFR and %TR of water.
Results
The functional viability of each IPK was assessed using
GFR and %TR of water, glucose and sodium. A kidney was
considered viable if the GFR and the %TR of water, glucose
and sodium were greater than 0.4 ml/min and greater than
80%, 90% and 85%, respectively, over the experimental period. For the single- and multiple-dose IPK studies, each of
these parameters was shown to remain relatively constant
with time, which is in keeping with the results of previous
studies from our laboratory (Mancinelli et al., 1995). No
significant differences were observed among the three groups
of single-dose IPKs for each of the functional parameters (P .
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Extraction solvent (6 ml of chloroform containing 20% n-butyl alcohol) was added to the buffered mixture, and the tube contents were
rotary-mixed at 33 rpm for 10 min and then centrifuged at 1800 3 g
for 10 min. After removal of the upper aqueous phase by aspiration,
5 ml of the organic phase was transferred to a 10-ml tube containing
0.05% sulfuric acid (200 ml), and this mixture was rotary-mixed (33
rpm, 10 min) and then centrifuged (1800 3 g, 10 min). A 150-ml
aliquot of the acid phase was transferred to a HPLC automatic
injector vial, and 100 ml was injected onto the HPLC column.
The HPLC system consisted of a LC-10AT pump, SIL-10A automatic injector, SCL-10A system controller, SPD-6A UV spectrophotometric detector (210 nm) and C-R6A Chromatopac integrator, all
from Shimadzu (Tokyo, Japan), and a Nova-pak C18, 4-mm RadialPak cartridge and Nova-Pak C18 Guard-Pak insert, both from Waters (Milford, MA). Mobile phase (70 mM potassium dihydrogen
orthophosphate buffer, pH 3, containing 1.5% acetonitrile and 1%
methanol) was delivered in single-pass mode to the column at a flow
rate of 1 ml/min. Typical retention times for normorphine and morphine were 4.9 and 6.2 min, respectively. The chromatographic run
time for each sample was 35 min because of the presence of lateeluting endogenous compounds. Calibration curves, ranging from
0.013 to 1.33 mM, were linear (r2 . 0.998), and repeat analysis of
quality-control samples containing morphine at concentrations of
0.027 mM, 0.27 mM and 1.06 mM indicated that the interday and
intraday accuracy and precision of the assay were within 11%.
The concentration of morphine in urine was determined by direct
injection of 100 ml of diluted urine (1:10 –1:5000) onto the HPLC
system described above. Calibration curves containing morphine
(0.026 –1.33 mM) in diluted urine (1:50) were linear (r2 . 0.996), and
repeat analysis of quality-control samples containing morphine at
concentrations of 0.135 mM, 0.54 mM and 1.06 mM indicated that the
interday and intraday accuracy and precision of the assay were
within 7%.
The potential role of the kidney in the formation of normorphine,
the internal standard, was assessed by testing for the presence of
normorphine in urine and perfusate samples that contained no internal standard. The possible presence of M3G and M6G in perfusate
and urine samples was assessed by using a validated HPLC assay
with UV detection involving solid-phase extraction of perfusate samples and direct injection of diluted urine samples according to a
modification of a previously reported method (Evans and Shanahan,
1995a).
Glucose concentrations in perfusate and urine were determined by
the glucose oxidase method, using a commercially available kit (Glucose Kit, Sigma Diagnostics, Sigma Chemical Co., St. Louis, MO).
Sodium concentrations in perfusate and urine samples were determined by atomic absorbance spectrophotometry (Varian Techtron
Atomic Absorbance Spectrophotometer, model no. AA6, Melbourne,
Victoria, Australia).
1997
Renal Disposition of Morphine
TABLE 1
Fraction of morphine unbound in IPK perfusate
Data presented as mean 6 S.D.
A. Morphine concentration covering a 100-fold range representative of the singledose IPK studies.
Treatment
Low
Medium
High
Morphine Concentration (mM)
fu
0.1
1
10
0.727 6 0.068
0.727 6 0.012
0.778 6 0.009
B. Morphine concentration covering a 1000-fold range representative of the multiple-dose IPK studies.
Treatment
Low
Medium
High
Extra High
Morphine Concentration (mM)
fu
0.2
2
20
200
0.722 6 0.073
0.743 6 0.020
0.773 6 0.004
0.719 6 0.025
Fig. 1. Concentration of morphine in perfusate vs. time profile for
single-dose studies; low (E; n 5 4), medium (h; n 5 4) and high (‚; n 5
4). Data are presented as mean and S.D.
profiles for the multiple-dose IPK studies are shown in figure
3. As expected, a 10-fold increase in perfusate concentration
was observed after each dose of morphine. CLM
R was 1 to 2
ml/min (fig. 4A) and was greater than fu z GFR during each
M
time interval (fig. 4B). Both CLM
R (P 5 .003) and the CLR /fu z
GFR ratio (P 5 .010) were lower at a concentration of 0.2 mM
(5–15 min) compared with the medium (20 –30 min), high
(35– 45 min) and extra-high (50 – 60 min) morphine concentrations.
A highly significant positive relationship was observed beM
tween CLM
R and GFR and between CLR 2 fu z GFR and GFR
for both the single-dose and the multiple-dose IPK studies
(table 3). Significant positive relationships were also observed between CLM
R and UFR or %TR of water and between
CLM
R 2 fu z GFR and UFR or %TR of water for the single-dose
IPK studies.
Discussion
Previous studies in the rat IPK have shown that morphine
undergoes renal tubular secretion (Ratcliffe et al., 1985; van
Crugten et al., 1991), and it has been postulated that morphine also undergoes carrier-mediated reabsorption (van
Crugten et al., 1991). Until now, however, there has been no
information about the effect of increasing concentration on
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.05). Therefore, morphine had no effect on the functional
viability of the IPK.
There was no evidence for the presence of the oxidative
metabolite normorphine or of the conjugative metabolites
M3G and M6G in perfusate and urine samples, as determined by two individual HPLC methods. With the sensitivity
limits of the respective assays for normorphine and M3G and
M6G, it would have been possible to detect 0.1% conversion of
morphine to normorphine over a 90-min perfusion and a
0.35% conversion of morphine to either M3G or M6G. Because normorphine was not formed by the kidney, its use as
an internal standard in the analytical method was not compromised.
Binding of morphine to protein in perfusate was low and
independent of morphine concentration over a 1000-fold
range, P . .05, (table 1). The average fu values used for the
calculation of CLM
R /fu z GFR ratios were 0.744 and 0.739 for
the single- and multiple-dose IPK studies, respectively.
The mean concentration of morphine in perfusate vs. time
profiles for the three groups of single-dose IPK studies are
presented semilogarithmically in figure 1. Profiles were biphasic in nature with an initial rapid distribution phase,
lasting for approximately 5 min, followed by a slower elimination phase. The terminal elimination phases for the three
treatment groups were observed to be parallel. Data on the
renal excretory clearance of morphine in each urine collection
interval from the single-dose experiments are shown in figure 2, and pharmacokinetic parameters for these kidneys are
presented in table 2. CLM
R was 0.8 to 2 ml/min (fig. 2A) and
was greater than fu z GFR during each time interval (fig. 2B).
There were no significant differences among the three groups
M
(P . .05) in the CLM
R /fu z GFR ratio and CLR estimates for
M
each time period and in CLR(0
.
However,
there was a
–90)
M
significant decrease with time in CLM
and
the
CL
R
R /fu z GFR
ratio for the low-dose and medium-dose groups. For each of
the three groups, there was no statistically significant differM
ence between the two clearance estimates, CLR(0
–90) and
M
CL (P . .05); however, upon combining the data for the
three groups we detected a small but significant difference
(P 5 .02) between these two clearance estimates. The mean
VM
K values ranged between 25 and 31 ml/g kidney weight, and
there were no significant differences among the three groups
(P . .05) for VM
K and T1/2.
The mean concentration of morphine in perfusate vs. time
1521
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Shanahan et al.
Vol. 282
TABLE 2
Pharmacokinetic parameters for morphine disposition observed
in single-dose IPK studies, for the low, medium and high
morphine concentrations
Data presented as mean 6 SD.
Concentration of Morphine in Perfusate
Parameter
T1/2 (min)
M
CLR(0–90)
(ml/min)
CLM
(ml/min)
VM
K (ml/g
kidney)
Statistical
Significance
Low (0.2 mM)
Medium (2 mM)
High (20 mM)
121 6 75
1.24 6 0.36
71 6 17
1.51 6 0.29
103 6 64
1.36 6 0.78
P 5 .491
P 5 .759
1.35 6 0.64
1.88 6 0.47
1.70 6 1.21
P 5 .676
31 6 3
25 6 9
26 6 7
P 5 .408
the renal excretory clearance of morphine and the relative
contribution of the various renal clearance mechanisms.
Moreover, there is controversy in the literature regarding the
possible role of the rat kidney in the metabolism of morphine.
Hence, the aims of the present studies in the IPK were to
investigate the effect of alterations in morphine concentration on the renal disposition of the drug and to resolve the
uncertainty about the contribution of the rat kidney to the
metabolism of morphine.
The rat IPK is a suitable model for studying the renal
handling of drugs, because tubular transport systems, in
particular those involved in secretion, remain viable while
making possible the study of renal disposition in the absence
of confounding factors such as extrarenal metabolism (Bekersky, 1983). Reabsorption of water, which occurs along the
entire tubule, is decreased in the IPK, resulting in a diminished driving force for passive reabsorption of organic solutes, whereas carrier-mediated reabsorption, which is confined largely to the proximal tubule, is well preserved
(Maack, 1980, 1986; Besseghir and Roch-Ramel, 1987).
The kidney is known to be involved in the metabolism of
xenobiotics (Jones et al., 1979; Anders, 1980). Glucuronidative metabolism of 1-naphthol (Redegeld et al., 1988) and
oxidative metabolism of meperidine (Acara et al., 1981) have
been observed previously in the rat IPK. The lack of normorphine, M3G and M6G in perfusate and urine samples collected in the present series of IPK studies indicates that the
rat kidney is not capable of metabolizing morphine to these
metabolites. Indeed, judging by the limit of quantification of
the respective assays, the degree of conversion of morphine to
normorphine, M3G or M6G must have been less than 0.4%.
The apparent absence of glucuronidation of morphine in the
IPK confirms the findings of Ratcliffe et al. (1985) and van
Crugten et al. (1991) in IPKs from unspecified and male
Hooded-Wistar rats, respectively, and is consistent with
studies performed with microsomes prepared from the kid-
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M
Fig. 2. CLM
R (panel A) and index of renal tubular transport, CLR /fu z GFR
(panel B) for the three groups of the single-dose studies; low (h; n 5 4),
medium (■; n 5 4) and high (u; n 5 4). Data are presented as mean and
S.D.
Fig. 3. Concentration of morphine in perfusate vs. time profile for
multiple-dose studies (n 5 6), 0.2 to 200 mM). Data are presented as
mean and S.D.
1997
Renal Disposition of Morphine
1523
TABLE 3
Bivariate regression analyses for the combined low, medium and
high groups of the single-dose IPK studies and multiple-dose
IPK studies
Statistical significance is represented as * P , .05, ** P , .01 and *** P , .001.
A. Single-dose IPK studies
Relationship
CLM
R
CLM
R
CLM
R
CLM
R
CLM
R
CLM
R
vs. GFR
vs. UFR
vs. %TR water
2 fu z GFR vs. GFR
2 fu z GFR vs. UFR
2 fu z GFR vs. %TR water
Regression Coefficient
Significance
0.747
0.441
0.283
0.658
0.400
0.238
***
***
**
***
***
*
B. Multiple-dose IPK studies
Relationship
M
Fig. 4. CLM
R (panel A) and index of renal tubular transport, CLR /fu z GFR
(panel B) for the multiple-dose studies (n 5 6) in the presence of
sequentially increasing morphine concentration (0.2–200 mM). Data are
presented as mean and S.D. Statistically significant difference observed between the low concentration (5–15 min) and each of the other
three concentrations of morphine, *P , .05 **P , .01.
ney of Fischer 344 rats (Rush et al., 1983; Rush and Hook,
1984).
Horton and Pollack (1991), using female Sprague-Dawley
rats, hypothesized a role for the kidney in the metabolism of
morphine after performing dispositional studies in intact
rats and rats in which the bile duct was cannulated and renal
pedicles were ligated. These investigators estimated that
renal metabolic clearance accounted for 28.5% of the total
systemic clearance of morphine. Their conclusion concerning
renal metabolism of morphine was based on a number of
assumptions, perhaps the most important being that the
hepatic clearance of morphine remained constant upon renal
ligation. Violation of this or any other of their assumptions
may explain their unsubstantiated finding on the role of the
kidney in morphine metabolism.
The study by Horton and Pollack (1991) involved the use of
female Sprague-Dawley rats, whereas the current studies
were performed using male Sprague-Dawley rats. Hence gender differences may explain the contrasting findings for the
vs. GFR
vs. UFR
vs. %TR water
2 fu z GFR vs. GFR
2 fu z GFR vs. UFR
2 fu z GFR vs. %TR water
Regression Coefficient
Significance
0.902
0.362
0.371
0.807
0.353
0.302
***
P 5 .0901
P 5 .0811
***
P 5 .0984
P 5 .1614
renal metabolism of morphine. Interestingly, Chhabra and
Fouts (1974) observed a 2-fold greater rate of glucuronidation
of p-nitrophenol in female rat renal microsomes of the CD
strain than in male rat renal microsomes. This observation
was supported by Rush et al. (1983) for the glucuronidation of
p-nitrophenol by renal microsomes from female and male
Fischer 344 rats. However, Rush et al. (1983) did not observe
glucuronidation of morphine in renal microsomes from either
female or male Fischer 344 rats.
Using the isolated perfused liver preparation, we have
previously shown that normorphine is formed in significant
quantities in the male rat, but not the female rat (Evans and
Shanahan, 1995b). However, oxidative metabolism of morphine in the kidney has not been assessed previously. In the
current study, there was no evidence for the metabolism of
morphine to normorphine in the male rat IPK.
Further evidence for the lack of a significant role for the rat
kidney in morphine metabolism comes from the finding, in
each of the three groups of the single-dose studies, that
M
M
CLR(0
(P . .05). It is
–90) was similar in magnitude to CL
interesting to note, however, that when the data from all
three groups were combined, CLM was significantly greater
M
than CLR(0
–90) (P , .05), and this may suggest that a metabolic route other than those assessed in the present study
plays a role in morphine metabolism. An alternative explanation, however, may be that extensive tissue uptake of
morphine, with subsequent slow release into perfusate, led to
an overestimate of the total clearance value calculated as
dose divided by AUC`
0 . Previous studies in the chicken kidney
(May et al., 1967; Watrous et al., 1970) have shown that
morphine undergoes renal metabolism to morphine ethereal
sulfate; however, this pathway for metabolism of morphine is
minimal in humans (Yeh et al., 1977) and rats (Smith et al.,
1973).
In the present study, the binding of morphine to albumin
in perfusate was low (table 1) and comparable to that observed in human plasma (Olsen, 1974; Milne et al., 1992) and
IPK perfusate (van Crugten et al., 1991). The fu of morphine
was independent of morphine concentration over a 1000-fold
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CLM
R
CLM
R
CLM
R
CLM
R
CLM
R
CLM
R
1524
Shanahan et al.
model (Boom et al., 1994). Saturation of cimetidine tubular
transport is possible because cimetidine has a relatively high
affinity for secretory transport in the rat kidney (Boom et al.,
1994). In the present study, the CLM
R /fu z GFR ratio for
morphine remained constant over the concentration range
0.2 to 20 mM used in the single-dose experiments. In the
multiple-dose study, the CLM
R /fu z GFR ratio was marginally
but significantly higher at concentrations of 2, 20 and 200
mM compared with that at 0.2 mM. The small change in the
ratio cannot be explained by saturation of renal tubular
secretion, because this would have resulted in a decrease in
renal clearance. Hence the results of the present studies
indicate that the renal secretion of morphine was not saturated over the 1000-fold range of concentrations investigated.
The lack of saturation suggests that morphine, like some
other organic cations, has a low affinity for the transport
system(s) involved in renal secretion. Transport of the organic cation N1-methylnicotinamide across the contraluminal renal tubular membrane occurs with a Km of 540 mM,
whereas movement of N-methylphenylpyridinium from the
lumen into the cell occurs with a Km of 200 mM in the rat
proximal tubule (Ullrich, 1994). Ullrich and Rumrich (1995),
using the stop-flow microperfusion method in the rat proximal tubule, determined a somewhat lower affinity for morphine transport, with Ki values of 780 mM and 1150 mM for
the inhibition of the transport of N1-methylnicotinamide and
N-methylphenylpyridinium across the contraluminal and luminal membranes, respectively.
van Crugten et al. (1991) suggested that morphine undergoes carrier-mediated tubular reabsorption in the rat kidney.
The basis of this hypothesis was an increase in the CLM
R /fu z
GFR ratio for morphine after the co-administration of M3G
and M6G to an IPK perfusion system. In the present study it
was not possible to quantitate the degree of net tubular
reabsorption, but reabsorption may have contributed to the
decrease observed in the CLM
R /fu z GFR ratio for the low and
medium groups of the single-dose studies as well as to the
slightly lower CLM
R /fu z GFR ratio observed for the 0.2 mM
morphine concentration in the multiple-dose studies.
A highly significant positive relationship was observed between the renal clearance of morphine and GFR (table 3),
which is not unexpected given that filtration is an important
component of renal clearance. Upon removal of the filtration
component (i.e., subtraction of fu z GFR from CLM
R ), a significant positive relationship with GFR remained, which suggests that the net renal tubular transport of morphine and
GFR are related. Possible reasons for this relationship are,
first, that tubular function and net secretory transport are
related to GFR and, second, that an increase in GFR produces an increase in UFR that indirectly results in a decrease
in the fractional reabsorption of morphine.
The CLM
R /fu z GFR ratio observed in the present study was
approximately half the corresponding ratio observed by van
Crugten et al. (1991), which indicates that in the present
study, either secretion was lower or reabsorption was more
extensive. Comparison of the %TR for water for the two
studies supports the latter of these two hypotheses. Thus, the
tubular reabsorption of water observed by van Crugten et al.
(1991) was approximately 50% less than that observed in the
current study. Hence the passive reabsorption of morphine,
which is driven by the concentration gradient created by the
reabsorption of water, may have been higher in the current
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range, a result comparable to findings in human plasma
(Olsen, 1974). It is not surprising that the fu of morphine was
concentration-independent, because the concentration of
BSA ('950 mM) utilized in the IPK perfusate was nearly
5-fold greater than that of the highest morphine concentration studied (200 mM). Hence the concentration of the binding
sites exceeded that of the ligand, and under these circumstances the unbound fraction would be expected to be independent of ligand concentration.
The disposition of morphine in the IPK system was characterized by accumulation in the kidney and by a renal clearance that involves net tubular secretion. Morphine was
shown to distribute rapidly and extensively into kidney tissue with a resultant VM
K of approximately 30 ml/g kidney
tissue, which was independent of perfusate morphine concentration over the 100-fold range used in the single-dose studies (table 2). A large volume of distribution of morphine was
previously observed in rats (11–27 l/kg) (Iwamoto and Klaassen, 1977; Bhargava et al., 1991), which supports the large
VM
K observed in the present study. Extensive uptake and
distribution within the kidney were also observed in a study
by Mullis et al. (1979) in which radiolabeled morphine was
administered to rats; uptake of morphine by the kidney was
substantially greater than that by other organs, including
the liver. In addition, extensive uptake of morphine by kidney slices from the dog (Hug, 1967) and mouse (Teller et al.,
1976) has been observed, and the findings are consistent with
the involvement of carrier-mediated mechanisms of renal
uptake.
The CLM
R values (figs. 2A; 4A) observed in the present
study are similar to values reported from previous studies in
the rat IPK (van Crugten et al., 1991) and the intact rat
(Horton and Pollack, 1991). For all time intervals in each
kidney in the single- and multiple-dose studies, the CLM
R /fu z
GFR ratio was greater than unity, with no ratio less than
1.64, a result that indicates net renal tubular secretion of
morphine. This finding is consistent with the renal handling
index observed for morphine in vivo in humans (Milne et al.,
1992; Somogyi et al., 1993) and sheep (Milne et al., 1995) and
in previous studies using the rat IPK (Ratcliffe et al., 1985;
van Crugten et al., 1991).
In the kidney, separate transport systems exist for the
secretion of organic anions (Møller and Sheikh, 1983) and
cations (Rennick, 1981) across the contraluminal and luminal membranes of the proximal tubule cells, and each transport system has a broad and overlapping substrate specificity. Contraluminal organic cation transport is driven by an
electrical potential difference, whereas luminal organic cation transport occurs via electroneutral H1/organic cation
exchange (Holohan and Ross, 1980; Somogyi, 1987; Ullrich,
1994). P-glycoprotein exists in proximal cells (Dutt et al.,
1994), and this transporter has been shown to be involved in
the transport of morphine across the luminal membrane of
renal proximal tubule cells of mice and humans (Schinkel et
al., 1995).
Tubular secretion, like any other carrier-mediated transport event, has a limited capacity, so saturation of secretion
may occur. Cimetidine, which like morphine also exists as an
organic cation at physiological pH, undergoes active tubular
secretion that is saturated with increasing concentration of
cimetidine over the range of 8 to 793 mM in rats in vivo
(Weiner and Roth, 1981) and 10 to 40 mM in the rat IPK
Vol. 282
1997
studies. Morphine is a relatively lipophilic molecule with an
octanol to pH 7.4 phosphate buffer partition coefficient for
the unchanged species of approximately 6 (Milne et al., 1996);
hence it may be expected to be passively reabsorbed from
urine into the peritubular capillary.
In conclusion, the rat IPK did not mediate the glucuronidation or oxidative metabolism of morphine. The disposition of
morphine in the kidney involved tubular secretion and intracellular accumulation, both of which were unaffected by
changes in morphine concentration.
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Renal Disposition of Morphine