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Cortisol metabolism and excretion in the isolated perfused rat kidney
2167
621
CORTISOL
METABOLISM PERFUSED
AND RAT
EXCRETION
IN
THE
ISOLATED
KIDNEY.
G. REACR, ll. NAKANE, Y. NAKANE, C. AUZAN and P. CORVOL. INSERM, U-36, 17 Rue du Fer-8-Moulin,
75005 Paris (FRANCE).
ABSTRACT
Rec'd. 7-l-77.
The isolated perfused rat kidney allows a simultaneous kinetic study of both the renal metabolism and the urinary excretion of cortisol and its metabolites in the rat. In this system, cortisol was completely metabolized within 120 minutes. The main renal metabolites of cortisol (cortisone, 20 reduced cortisol and 20 reduced cortisone) were found in the recirculating perfusate and in urine. The formation of these metabolites was quantitatively evaluated and compared to a theoretical model.
INTRODUCTION
The renal clearance of a steroid hormone is the result of both metabolism and urinary excretion. It is difficult to study these two pathways by conventional methods. -In vitro studies, performed by incubation of slices or homogenates present an obvious difficulty : the functional integrity of the kidney is not preserved and the contribution of the urinary excretion to the renal clearance of the hormone cannot be evaluated. On the other hand, the presence of the liver makes interpretation of -in vivo studies on the renal metabolism of a steroid hormone difficult. The aim of this work was to study simultaneously the renal metabolism and the urinary ex-
VoZwne 30, Number 5
S
'EDEOXDI
November,
2977
S
TEIEOIDN
cretion of a steroid hormone in an isolated perfused rat kidney model. As cortisol is known to be metabolized
in kidney in many spe-
cies including the rat (1,2), this hormone was chosen for this investigation. Cortisol metabolism
in kidney was demonstrated to be
less complex than in liver : the main metabolites were cortisone, 20$-dihydro-cortisol
and 20g-dihydrocortisone
(1,2).
MATERIALS AND METHODS
Kidney perfusion The right kidney of male Wistar rats (350-450g) was perfused with blood-free Krebs-Ringer bicarbonate buffer containing 5 % of bovine serum-albumin (Armour Cohn Fraction V) and 5.5mmole glucose The perfusion apparatus and perfusion procedure were identical to those described by Bowman (3), with the exception of a modification of the circuit arrangement, as represented in Fig.1. The isolated kidney was suspended at the top of a multibulb glass condenser (A) with an arterial cannula (B), connected to a peristaltic pump (C)(Masterflex, Cole Parmer Instr. Co., USA). A mixture of 02 / CO2 (95 : 5) was hydrated with a humidifier column (D) and delivered into the lower portion of the condenser. Oxygenation due to the contact of the gas mixture with the perfusate passing down in the chamber was further accelerated by the application of a shunting circuit (E) in parallel with the main circuit. This modification also allowed a decrease in the mixing time of the whole system. The medium was thermostated at 37OC. Perfusion pressure was maintained between 65 and 85 mm Hg (manometer F). Fig. 1. See text for explanations. The operative procedure was as described by Bowman (3). Briefly after the anesthesia, with sodium pentobarbital, the right ureter was catheterized and the renal artery was cannulated without interrupting the renal blood flow. The perfusate was initially maintained by a temporary gravity flow
system until the kidney was isolated and placed in the oxygenator colon. Then the recirculating system was started. The adrenal gland was carefully removed. The perfusate volume was 82-85 ml and was kept constant with prepared solutions during the experiment. Since losses of perfusate occured due to serial sampling, compensation was performed with the same original Krebs-Ringer solution. Urinary losses were caapensated as described elsewhere (4). Steroid studies Materials _---I--1,2 3H-cortisol (40 Ci/mmole), 4-14C-cortisol (54mCi/usaole) and 4-14C-cortisone (58 mCi/mnole) were obtained from Amersham Radiochemica1 Center. These compounds were diluted in ethanol and stored at -15'C in liquid form. The purity of the solutions was checked by paper chromatography. Stable compounds 20S-dihydrocortisoland 208-dihydrocortisonewere obtained from Steraloids. Their purity was checked by gas liquid chromatography. Chemical and solvents were of analytical grade. Methods ---___Recirculating of cortisol in the apparatus without kidney. --_----_---- _____________________ ____________________ In a preliminary experiment, it was verified that 3l+cortisol was not adsorbed or metabolized during two hours of circulation in the apparatus without kidney. Cortisd metabolism and excretion study--------_---_---in the isolated perfused "'r"-""-"""'-""""""--"-' _______ rat kidney. --------All studies were done after 20 minutes of equilibrium of the system with kidney. Then I uCi 3H-cortisol, dissolved in 3 ml of perfusate was injected in the recirculatingmedium. Urine was then collected for serial periods of ten minutes and the perfueate was sampled at the middle of each time period. For each sample, an aliquot was dissolved in Unisolve scintillation liquid (Koch-Light) and counted. In another aliquot, 2000 dpm of 14C-labelled cortisol and cortisone were added for recovery calculations. The perfusate and urine samples were then extracted with ten volumes of ethyl acetate, and the extract was submitted to paper chromatography in a Bush system (Benzene : Methanol : Water : Ethyl Acetate, I : 1 : 1 : 0.1) for 5 hours. In such a system, cortisol and cortisone were easily and completely separated. The radioactivity corresponding to cortisol and cortisone present in each successive sample was calculated and the internal losses corrected. Overall recovery was about 60 X. Cortisol and its metabolites were also calculated in percent of the total radioactivity. Organic extracts were counted in Toluene-Popop-PPO. Gas liquid chranatogrqhy was used for identification of the "'7" two main rne~a~;;iTSes;;f-cortisol. In order to obtain a suitable deri-
vative for gas liquid chromatography analysis, the residue was dissolved in 0.5 ml of a mixture methyloxime hydrochloride pyridine (1 mg/ ml). This solution was allowed to stand at room temperature overnight. After evaporation, the silylation was carried out by diluting the residue with TRISIL "TBT" (Pierce Chemical CO.). This reduction was performed at 60°C overnight. The TM Si derivative was then analyzed in G.L.C. with the following characteristics : Column : 1 % SE 30 ; length : 2 m ; oven to : 240°C ; injection part to : 285'C ; Detector to : 285 'C ; N2 was used as carrier gas (Intersmat IGC 15). Kidney function analysis _____ ______________ ___ : yt omerular filtration rate (GFR) was C-polyethyleneglycol (M.W. 4000). evaluated as the clearance of The renal perfusate flow was evaluated with the flow-meter (G on Fig. 1). Na concentration in perfusate and urine were determined spectrophotometrically. Statistical analysis -f--:----'l-'~-~ -a- : Student's t test was employed to determine statistical significance. The following __-____--_____ trivial names have been used in this paper : ____________ Cortisol (F) : 118, 17a, 21-trihydroxypregn-4-ene-3, 20-dione. Cortisone (E) : 17a, 21-dihydroxypregn-4-ene-3, II, 20-trione. Aldosterone : 18, Il-hemiacetal of 118, 21-dihydroxy-3, 20-dioxo-4pregnen-18-al. 208-dihydro cortisol : 118, 17a, 208, 21-tetrahydroxypregn-4-ene3-one. 208-dihydro cortisone : 17a, 208, 21-trihydroxypregn-4-ene-3, IIdione. B cortol : 5B-pregnane-Za, llf3, 17, 208, 21-pent01 8 cortolone : 3c(, 17ct, 208, 21-tetrahydroxy-58-pregnan-U-one As no investigation was attempted for identifying the 20~ and 208 epimers of 20 reduced cortisol and cortisone, the terms "20 reduced cortisol" and "20 reduced cortisone" will be used. abbreviated as DHF and DHE in figures. RESULTS
Functional characteristics
of the isolated perfused rat kidney.
The main features of the kidney function during the 120 minutes perfusion are indicated in Table 1. It can be seen that glomerular filtration rate (GFR) and renal perfusate flow (RPF) remained constant during the entire study. Na reabsorption decreased slowly during the perfusion. Results are expressed by g of the contralateral kidney, as discussed by Bowman
(3).
S
?rDEOXDrn
625
TABLE I Minutes
Perfusion pressure mm Ug
GFR ml/min/g
RPF mllminfg
X Na Reabsorption
O-30
73.3+10.3
0.39+0.27
25.9L3.7
9029
30-60
75.1+11.8
0.4620.24
24.523.7
87210
60-90
77.2+11.8
0.42+0.18
23.7L3.6
85+13
go-120
79.3+11.1
0.31+0.17
23.1~3.5
83+15
Data are expressed as mean and standard deviation (n = 6). Total radioactivity handling by the isolated perfused rat kidney. The disappearance of the total radioactivity in the perfusate and its appearance in urine is shown in Fig. 2. After 120 minutes of perfusion, about 77 % of the initial radioactivitywas still present in the recirculatingmedium, whereas 17 % was found in urine.
60
120 min
Total radioactivity in perfusate and urine, vs time of perfusion. Urinary results are expressed cumulatively.
S
626
-JFDEOID=
A positive correlation was found between the total urinary radioactivity excreted during the 120 minutes perfusion and the mean diuresis of each kidney (r = 0.85, n = 6, p < 0.05).
Cortisol metabolism in the isolated perfused rat kidney. An extensive cortisol metabolism was observed in the isolated perfused rat kidney. There was a slow decrease in total radioactivity of the perfusate, but %I-cortisol decreased rapidly with very little remaining at the end of the perfusion, as shown in Fig. 3. PERFUSATE
dyz I
ld
.
60
120min
3 Fig.3. Concentration of 3H-cortisol (-c->, H-cortisone (-0-), polar %metabolites (+), and total radioactivity (-#--) in the perfusate of an individual experiment. Open squares represent the sum of the radioactivity of the three kinds of steroids.
s
627
TIEIEOXDI
The radioactivity still present in the medium after 120 minutes of perfusion was therefore represented by other metabolites than cortisone. These metabolites were more polar than cortisol in the Bush chromatographic system. At each time period, the calculated sum of the % -cortisol, cortisone and polar metabolites was close to the total radioactivity, indicating that these metabolites represented the main metabolites of cortisol in this system. Identification of cortisol metabolites in isolated perfused rat kidney. Cortisol and its metabolites were
2X X103
‘I
directly extracted by an organic solvent from the medium, without a prealable hydrolysis, suggesting that they circulate in an unconjugated form. The chromatographicpattern of perfusate at the end of the perfusion is shown in Fig. 4. Five tritiated peaks were detected : two main peaks, I and II, more polar than cortisol ; the peaks (III and IV) of unmetabolized cortisol and cortisone ; a small 6.F
peak (V) less polar than cortisone. Peaks I and 11 were allowed to
‘6.E
I
I
III
IV
3
Fig. 4. Chromatogram of an aliquot of perfusate, at the end of the perfusion. 14C-F and 14C-E represent the migration of the 14C labeled corresponding steroids.
front
S
TRROID6
migrate in the same system for 12 hours in comparison with the stable standards 20B-dihydro cortisol and 20S-dihydro cortisone. Under these conditions metabolites ty was respectively
I and II were well separated and their mobili-
identical to that of 20S-dihydrocortisol
and
20S-dihydrocortisone. Further identification of stable compounds I and II by gas liquid chromatography
(G.L.C.) was performed as follows
from an adrenalectomized
: a kidney obtained
rat (in order to avoid a contamination by
endogenous steroids) permitted the preparation of stable compounds I and II from the perfusion of 0.5 mg of stable cortisol. These two compounds were first purified by paper chromatography as above and then submitted to G.L.C. Their retention time was compared to those of the corresponding
standards. Each compound exhibited a single peak
on G.L.C., corresponding
to the retention time of 20S-dihydrocortisol
for peak I and 20S-dihydrocortisone
for peak II. The same metabolic
pattern was found in urine.
Short term kinetic study of the metabolites
apparition in perfusate
and in urine. The radioactivity corresponding dihydrocortisol
to cortisol, cortisone, 2Of3-
and 206-dihydrocortisone
was expressed in percent of
the total radioactivity present in perfusate and in urine at each time point. Fig. 5 shows a typical pattern of the different metabolites in perfusate and in urine. At the end of the perfusion, 20reduced cortisol and 20-reduced cortisone represented respectively 57.5 + 3.2 and 39.9 -+ 1.4 % of the total radioactivity present in the perfusate
(mean -+ S.D., n = 4).
s
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629
Fig. 5. Concentration of cortisol and its metabolites in perfusate and urine of an individual experiment. Results are expressed in percent of total radioactivity for each time.
F. E o DHF .( DHE o
Estimation of the cortisol metabolites formation. At each time period, the perfusate radioactivity and the cumulative urine radioactivity corresponding to each steroid were added. The results are expressed as a percent of the total radioactivity (perfusate and urine) present at each time. This allowed the comparison of the experimental data to a theoretical model of the biotransformation of cortisol as represented by : Cortisol
kl
k3 I 20 reduced cortisol
l
Cortisone
k2 I 20 reduced cortisone
%c
630
TDEOIDCI
theoretical
. . . .._......I
*...
experimental
Fig. 6. Comparison of theoretical $*-,-.-,-} and experimental ) data. Theoretical curves are obtained from the (equation shown in Appendix. where k
1' k2'
k3 represent the rate constants, as discussed by
Vinson and Whitehouse (5). Theoretical curves of formation of cortisol metabolites are shown in Fig. 6. The rate constants found were kl
= O.O18/minute, k2 = 0.200/minute, kS = 0.052lminute.
DISCDSSION
The determination of a renal steroid metabolic clearance rate in vivo is complicated by the presence of other organs able to meta-bolize the steroid, such as the liver. It involves renal artery and vein catheterization and serial blood sampling which are difficult in vitro experiments may to perform in small laboratory animals. The -present an alternative method for studying steroid metabolism. Incubation of radioactive steroids with kidney slices, minces or homoge-
s
WmEOxDm
631
nates have been used by many authors (1, 2). However, all these techniques, in which the functional integrity of the kidney is not preserved, do not permit thestudy of the renal excretion of the hormone. Moreover, the accumulation of the metabolites formed during the incubation could interfere with the activity of the steroid metabolizing enzymes. It therefore seemed that an isolated perfused kidney would be a convenient model for studying both steroid renal metabolism and excretion. The isolated perfused rat kidney used in this study derived from that described by Bowman (3). In this system, a semi-synthetic medium containing no blood or plasma was perfused. The functional characteristicsof the present system has been shown to work successfully, although there was a slight decrease in glaaerular filtration rate and in sodium reabsorption after two hours. In this model, an extensive cortisol metabolism could be easily demonstrated. Cortisol almost completely disappeared in the medium within 90 minutes after a half-life of IO-15 minutes. An important metabolism of cortisol was also demonstrated by Nicholas and Kim (6) in an isolated perfused rat lung. Thus isolated perfused organs,,in which metabolism is greater than in conventional_in vitro methods, are convenient for studying steroid metabolism in a given tissue. In the isolated perfused rat kidney, cortisol was metabolized into cortisone and two polar metabolites, which have the same mobility on paper and on gas liquid chromatography than authentic 206-dihydrocortisol and 20g-dihydrocortisonerespectively, although no further identificationwas attempted, i.e. the separation between the 20a
and 208
epimers.
However, these results, on the nature of the main metabolites of# cortisol in kidney, are in good agreement with the previous data reported -in vitro in the rat. Using slices, minces, or homogenates, Mahesh 3 (1) found that H-cortisol was metabolized to cortisone, 20@-dihydrocortisol and 20@dihydrocortisone. However, the percent of conversion of cortisol to its metabolites differed widely according to the -in vitro method used. 'Inman, the formation of cortisone, 20@-dihydro was also shown by Jenkins (Z), by cortisol and 20f3-dihydrocortisone in vitro slices incubation. Only the @form was found. In dogs, 20 B-dihydrocortisol and 20@-dihydrocortisonehave also been identified -in vivo in urine and are the only cortisol metabolites found when the liver has been removed, indicating their extra-hepatic origin (7). Moreover, from the present study, it appeared that the rat kidney does not conjugate -in vivo cortisol and its metabolites. The absence of glucuroconjugationhas also been shown by Mahesh (1) and -in vivo for aldosterone, using the same isolated perfused rat kidney model (4). The isolated perfused rat kidney also permits a metabolic shortterm kinetic study . The kinetic rate constants can be easily determined and the following schema of the cortisol metabolism in kidney is proposed, from the data expressed in Fig. 6. Cortisol
b-D
20 reduced cortisol
Cortisone
20 reduced cortisone
As shown here, the 20-reduction of cortisone appears to be faster than
s
TBEOXDI
633
that of cortisol. Finally, the renal handling of cortisol was studied with the use of this model. The urinary excretion of cortisol was found to be diuresis dependent as shown _in vivo in animals (8) and in man (9). As it is known that cortisol is submitted to a filtration-reabsorption process(lO), the influence of the diuresis on the urinary excretion of these steroids could be explained if one assumed that reabsorption decreases when diuresis increases. It should be noted that the relative concentration of 20-reduced cortisol was much higher in urine than in perfusate in the first periods of the perfusion. Since this compound is formed in the kidney and is recirculating in the perfusate, it is possible that the metabolite is more actively filtered and/or less reabsorbed than cortisol. This would explain why this metabolite was more rapidly excreted than cortisol. Alternatively, this compound may be synthetized in the kidney from cortisol, directly eliminated in urine and partially reabsorbed. Thus,a fraction of this metabolite may be directly excreted in urine without a recirculation in the perfusate. 20g-dihydrocortisoland 20B-dihydrocortisonehave been identified in human (11) and dog (12) urine. The origin of these compounds may he in the liver, although they are metabolized into S-cortol and S-cortolone (13). From our experiments, it is suggested that a fraction of these urinary compounds is formed in the kidney, then directly excreted in urine, escaping the hepatic metabolism. The isolated perfused rat kidney offers several advantages. However, the results obtained by the use of this model should he
S
634
TIlEOXDb
compared with true -in vivo studies since steroid metabolism by the kidney in vivo depends on other factors, such as protein binding. The isolated perfused rat kidney may be a convenient model for studying the influence of such extra-renal factors on the renal handling of steroid hormones.
APPENDIX Consider the model :
k3
[
k'
'1
D
k2
C
The differential equations are as follows : dA - -(kl + k3)A zdB= dt
kB-kC 1
dD dt
kA3
and C = 1 - (A+B+D)
2
These equations integrate into (if A0 = 1, B
= Co = Do = 0): 0
A
B=
=
e
-0y
+
k3)t
k, + k?
(e -(kl+k3)t
_ e -k2t )
k2 - (k,+k3)
D=
k3
(1 - e -(kl+k3)t
kl+k3 ACKNOWLEDGMENTS This work has been supported by grants from Institut National de la Santd et de la Recherche Mddicale (Contrat no 744093-5).
S
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635
REFBRENCES
1. 2. 3. 4.
Mahesh, V.B. and Ulrich,F.,J. Biol. Chem. 235, 356 (1960) Jenkins, J.S., J. Endocrinol. 34, 151 (196r Bowman, R.H., J. Biol. Chem. 245, 1604 (1970) Nakane, H., Nakan6, Y., Rgach, G., Corvol, P. and MBnard,J., Am. J. Physiol., in press. 5. Vinson, G.P. and Whitehouse, B.J., In Advances in Steroid Bioche? mistry and Pharmacology, i, 163, Academic Press, New York and London (1970). 6. Nicholas, T.E., and Kim, P.A., Steroids, 25, 387 (1975) 7. Gold, N.I., J. Biol. Chem. 236, 1930 (196n 8. Kalant, N., Am. J. Physiol. 132, 503 (1955) 9. Hatfield, C.B., and Schuster,., J. Endocrinol. 2, 262 (1959) 10. Beisel, W.R., Di Raimondo, V.C. and Forsham, P.H., Ann. Int. Med. 2, 641 (1964) 11. Holness, N.J., Lunnon, J.B., and Gray, C.H., J. Endocrinol. 14, 136 (1956) 12. Gold, N.I., J. Biol. Chem. 236, 1924 (1961) 13. Bradlow, H.L., Fukushima, D.K., Zumoff, B., Hellman, L. and Gallagher, T.F., J. Clin. Endocr. Metab. 22, 748 (1962).
621
CORTISOL
METABOLISM PERFUSED
AND RAT
EXCRETION
IN
THE
ISOLATED
KIDNEY.
G. REACR, ll. NAKANE, Y. NAKANE, C. AUZAN and P. CORVOL. INSERM, U-36, 17 Rue du Fer-8-Moulin,
75005 Paris (FRANCE).
ABSTRACT
Rec'd. 7-l-77.
The isolated perfused rat kidney allows a simultaneous kinetic study of both the renal metabolism and the urinary excretion of cortisol and its metabolites in the rat. In this system, cortisol was completely metabolized within 120 minutes. The main renal metabolites of cortisol (cortisone, 20 reduced cortisol and 20 reduced cortisone) were found in the recirculating perfusate and in urine. The formation of these metabolites was quantitatively evaluated and compared to a theoretical model.
INTRODUCTION
The renal clearance of a steroid hormone is the result of both metabolism and urinary excretion. It is difficult to study these two pathways by conventional methods. -In vitro studies, performed by incubation of slices or homogenates present an obvious difficulty : the functional integrity of the kidney is not preserved and the contribution of the urinary excretion to the renal clearance of the hormone cannot be evaluated. On the other hand, the presence of the liver makes interpretation of -in vivo studies on the renal metabolism of a steroid hormone difficult. The aim of this work was to study simultaneously the renal metabolism and the urinary ex-
VoZwne 30, Number 5
S
'EDEOXDI
November,
2977
S
TEIEOIDN
cretion of a steroid hormone in an isolated perfused rat kidney model. As cortisol is known to be metabolized
in kidney in many spe-
cies including the rat (1,2), this hormone was chosen for this investigation. Cortisol metabolism
in kidney was demonstrated to be
less complex than in liver : the main metabolites were cortisone, 20$-dihydro-cortisol
and 20g-dihydrocortisone
(1,2).
MATERIALS AND METHODS
Kidney perfusion The right kidney of male Wistar rats (350-450g) was perfused with blood-free Krebs-Ringer bicarbonate buffer containing 5 % of bovine serum-albumin (Armour Cohn Fraction V) and 5.5mmole glucose The perfusion apparatus and perfusion procedure were identical to those described by Bowman (3), with the exception of a modification of the circuit arrangement, as represented in Fig.1. The isolated kidney was suspended at the top of a multibulb glass condenser (A) with an arterial cannula (B), connected to a peristaltic pump (C)(Masterflex, Cole Parmer Instr. Co., USA). A mixture of 02 / CO2 (95 : 5) was hydrated with a humidifier column (D) and delivered into the lower portion of the condenser. Oxygenation due to the contact of the gas mixture with the perfusate passing down in the chamber was further accelerated by the application of a shunting circuit (E) in parallel with the main circuit. This modification also allowed a decrease in the mixing time of the whole system. The medium was thermostated at 37OC. Perfusion pressure was maintained between 65 and 85 mm Hg (manometer F). Fig. 1. See text for explanations. The operative procedure was as described by Bowman (3). Briefly after the anesthesia, with sodium pentobarbital, the right ureter was catheterized and the renal artery was cannulated without interrupting the renal blood flow. The perfusate was initially maintained by a temporary gravity flow
system until the kidney was isolated and placed in the oxygenator colon. Then the recirculating system was started. The adrenal gland was carefully removed. The perfusate volume was 82-85 ml and was kept constant with prepared solutions during the experiment. Since losses of perfusate occured due to serial sampling, compensation was performed with the same original Krebs-Ringer solution. Urinary losses were caapensated as described elsewhere (4). Steroid studies Materials _---I--1,2 3H-cortisol (40 Ci/mmole), 4-14C-cortisol (54mCi/usaole) and 4-14C-cortisone (58 mCi/mnole) were obtained from Amersham Radiochemica1 Center. These compounds were diluted in ethanol and stored at -15'C in liquid form. The purity of the solutions was checked by paper chromatography. Stable compounds 20S-dihydrocortisoland 208-dihydrocortisonewere obtained from Steraloids. Their purity was checked by gas liquid chromatography. Chemical and solvents were of analytical grade. Methods ---___Recirculating of cortisol in the apparatus without kidney. --_----_---- _____________________ ____________________ In a preliminary experiment, it was verified that 3l+cortisol was not adsorbed or metabolized during two hours of circulation in the apparatus without kidney. Cortisd metabolism and excretion study--------_---_---in the isolated perfused "'r"-""-"""'-""""""--"-' _______ rat kidney. --------All studies were done after 20 minutes of equilibrium of the system with kidney. Then I uCi 3H-cortisol, dissolved in 3 ml of perfusate was injected in the recirculatingmedium. Urine was then collected for serial periods of ten minutes and the perfueate was sampled at the middle of each time period. For each sample, an aliquot was dissolved in Unisolve scintillation liquid (Koch-Light) and counted. In another aliquot, 2000 dpm of 14C-labelled cortisol and cortisone were added for recovery calculations. The perfusate and urine samples were then extracted with ten volumes of ethyl acetate, and the extract was submitted to paper chromatography in a Bush system (Benzene : Methanol : Water : Ethyl Acetate, I : 1 : 1 : 0.1) for 5 hours. In such a system, cortisol and cortisone were easily and completely separated. The radioactivity corresponding to cortisol and cortisone present in each successive sample was calculated and the internal losses corrected. Overall recovery was about 60 X. Cortisol and its metabolites were also calculated in percent of the total radioactivity. Organic extracts were counted in Toluene-Popop-PPO. Gas liquid chranatogrqhy was used for identification of the "'7" two main rne~a~;;iTSes;;f-cortisol. In order to obtain a suitable deri-
vative for gas liquid chromatography analysis, the residue was dissolved in 0.5 ml of a mixture methyloxime hydrochloride pyridine (1 mg/ ml). This solution was allowed to stand at room temperature overnight. After evaporation, the silylation was carried out by diluting the residue with TRISIL "TBT" (Pierce Chemical CO.). This reduction was performed at 60°C overnight. The TM Si derivative was then analyzed in G.L.C. with the following characteristics : Column : 1 % SE 30 ; length : 2 m ; oven to : 240°C ; injection part to : 285'C ; Detector to : 285 'C ; N2 was used as carrier gas (Intersmat IGC 15). Kidney function analysis _____ ______________ ___ : yt omerular filtration rate (GFR) was C-polyethyleneglycol (M.W. 4000). evaluated as the clearance of The renal perfusate flow was evaluated with the flow-meter (G on Fig. 1). Na concentration in perfusate and urine were determined spectrophotometrically. Statistical analysis -f--:----'l-'~-~ -a- : Student's t test was employed to determine statistical significance. The following __-____--_____ trivial names have been used in this paper : ____________ Cortisol (F) : 118, 17a, 21-trihydroxypregn-4-ene-3, 20-dione. Cortisone (E) : 17a, 21-dihydroxypregn-4-ene-3, II, 20-trione. Aldosterone : 18, Il-hemiacetal of 118, 21-dihydroxy-3, 20-dioxo-4pregnen-18-al. 208-dihydro cortisol : 118, 17a, 208, 21-tetrahydroxypregn-4-ene3-one. 208-dihydro cortisone : 17a, 208, 21-trihydroxypregn-4-ene-3, IIdione. B cortol : 5B-pregnane-Za, llf3, 17, 208, 21-pent01 8 cortolone : 3c(, 17ct, 208, 21-tetrahydroxy-58-pregnan-U-one As no investigation was attempted for identifying the 20~ and 208 epimers of 20 reduced cortisol and cortisone, the terms "20 reduced cortisol" and "20 reduced cortisone" will be used. abbreviated as DHF and DHE in figures. RESULTS
Functional characteristics
of the isolated perfused rat kidney.
The main features of the kidney function during the 120 minutes perfusion are indicated in Table 1. It can be seen that glomerular filtration rate (GFR) and renal perfusate flow (RPF) remained constant during the entire study. Na reabsorption decreased slowly during the perfusion. Results are expressed by g of the contralateral kidney, as discussed by Bowman
(3).
S
?rDEOXDrn
625
TABLE I Minutes
Perfusion pressure mm Ug
GFR ml/min/g
RPF mllminfg
X Na Reabsorption
O-30
73.3+10.3
0.39+0.27
25.9L3.7
9029
30-60
75.1+11.8
0.4620.24
24.523.7
87210
60-90
77.2+11.8
0.42+0.18
23.7L3.6
85+13
go-120
79.3+11.1
0.31+0.17
23.1~3.5
83+15
Data are expressed as mean and standard deviation (n = 6). Total radioactivity handling by the isolated perfused rat kidney. The disappearance of the total radioactivity in the perfusate and its appearance in urine is shown in Fig. 2. After 120 minutes of perfusion, about 77 % of the initial radioactivitywas still present in the recirculatingmedium, whereas 17 % was found in urine.
60
120 min
Total radioactivity in perfusate and urine, vs time of perfusion. Urinary results are expressed cumulatively.
S
626
-JFDEOID=
A positive correlation was found between the total urinary radioactivity excreted during the 120 minutes perfusion and the mean diuresis of each kidney (r = 0.85, n = 6, p < 0.05).
Cortisol metabolism in the isolated perfused rat kidney. An extensive cortisol metabolism was observed in the isolated perfused rat kidney. There was a slow decrease in total radioactivity of the perfusate, but %I-cortisol decreased rapidly with very little remaining at the end of the perfusion, as shown in Fig. 3. PERFUSATE
dyz I
ld
.
60
120min
3 Fig.3. Concentration of 3H-cortisol (-c->, H-cortisone (-0-), polar %metabolites (+), and total radioactivity (-#--) in the perfusate of an individual experiment. Open squares represent the sum of the radioactivity of the three kinds of steroids.
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TIEIEOXDI
The radioactivity still present in the medium after 120 minutes of perfusion was therefore represented by other metabolites than cortisone. These metabolites were more polar than cortisol in the Bush chromatographic system. At each time period, the calculated sum of the % -cortisol, cortisone and polar metabolites was close to the total radioactivity, indicating that these metabolites represented the main metabolites of cortisol in this system. Identification of cortisol metabolites in isolated perfused rat kidney. Cortisol and its metabolites were
2X X103
‘I
directly extracted by an organic solvent from the medium, without a prealable hydrolysis, suggesting that they circulate in an unconjugated form. The chromatographicpattern of perfusate at the end of the perfusion is shown in Fig. 4. Five tritiated peaks were detected : two main peaks, I and II, more polar than cortisol ; the peaks (III and IV) of unmetabolized cortisol and cortisone ; a small 6.F
peak (V) less polar than cortisone. Peaks I and 11 were allowed to
‘6.E
I
I
III
IV
3
Fig. 4. Chromatogram of an aliquot of perfusate, at the end of the perfusion. 14C-F and 14C-E represent the migration of the 14C labeled corresponding steroids.
front
S
TRROID6
migrate in the same system for 12 hours in comparison with the stable standards 20B-dihydro cortisol and 20S-dihydro cortisone. Under these conditions metabolites ty was respectively
I and II were well separated and their mobili-
identical to that of 20S-dihydrocortisol
and
20S-dihydrocortisone. Further identification of stable compounds I and II by gas liquid chromatography
(G.L.C.) was performed as follows
from an adrenalectomized
: a kidney obtained
rat (in order to avoid a contamination by
endogenous steroids) permitted the preparation of stable compounds I and II from the perfusion of 0.5 mg of stable cortisol. These two compounds were first purified by paper chromatography as above and then submitted to G.L.C. Their retention time was compared to those of the corresponding
standards. Each compound exhibited a single peak
on G.L.C., corresponding
to the retention time of 20S-dihydrocortisol
for peak I and 20S-dihydrocortisone
for peak II. The same metabolic
pattern was found in urine.
Short term kinetic study of the metabolites
apparition in perfusate
and in urine. The radioactivity corresponding dihydrocortisol
to cortisol, cortisone, 2Of3-
and 206-dihydrocortisone
was expressed in percent of
the total radioactivity present in perfusate and in urine at each time point. Fig. 5 shows a typical pattern of the different metabolites in perfusate and in urine. At the end of the perfusion, 20reduced cortisol and 20-reduced cortisone represented respectively 57.5 + 3.2 and 39.9 -+ 1.4 % of the total radioactivity present in the perfusate
(mean -+ S.D., n = 4).
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Fig. 5. Concentration of cortisol and its metabolites in perfusate and urine of an individual experiment. Results are expressed in percent of total radioactivity for each time.
F. E o DHF .( DHE o
Estimation of the cortisol metabolites formation. At each time period, the perfusate radioactivity and the cumulative urine radioactivity corresponding to each steroid were added. The results are expressed as a percent of the total radioactivity (perfusate and urine) present at each time. This allowed the comparison of the experimental data to a theoretical model of the biotransformation of cortisol as represented by : Cortisol
kl
k3 I 20 reduced cortisol
l
Cortisone
k2 I 20 reduced cortisone
%c
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TDEOIDCI
theoretical
. . . .._......I
*...
experimental
Fig. 6. Comparison of theoretical $*-,-.-,-} and experimental ) data. Theoretical curves are obtained from the (equation shown in Appendix. where k
1' k2'
k3 represent the rate constants, as discussed by
Vinson and Whitehouse (5). Theoretical curves of formation of cortisol metabolites are shown in Fig. 6. The rate constants found were kl
= O.O18/minute, k2 = 0.200/minute, kS = 0.052lminute.
DISCDSSION
The determination of a renal steroid metabolic clearance rate in vivo is complicated by the presence of other organs able to meta-bolize the steroid, such as the liver. It involves renal artery and vein catheterization and serial blood sampling which are difficult in vitro experiments may to perform in small laboratory animals. The -present an alternative method for studying steroid metabolism. Incubation of radioactive steroids with kidney slices, minces or homoge-
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WmEOxDm
631
nates have been used by many authors (1, 2). However, all these techniques, in which the functional integrity of the kidney is not preserved, do not permit thestudy of the renal excretion of the hormone. Moreover, the accumulation of the metabolites formed during the incubation could interfere with the activity of the steroid metabolizing enzymes. It therefore seemed that an isolated perfused kidney would be a convenient model for studying both steroid renal metabolism and excretion. The isolated perfused rat kidney used in this study derived from that described by Bowman (3). In this system, a semi-synthetic medium containing no blood or plasma was perfused. The functional characteristicsof the present system has been shown to work successfully, although there was a slight decrease in glaaerular filtration rate and in sodium reabsorption after two hours. In this model, an extensive cortisol metabolism could be easily demonstrated. Cortisol almost completely disappeared in the medium within 90 minutes after a half-life of IO-15 minutes. An important metabolism of cortisol was also demonstrated by Nicholas and Kim (6) in an isolated perfused rat lung. Thus isolated perfused organs,,in which metabolism is greater than in conventional_in vitro methods, are convenient for studying steroid metabolism in a given tissue. In the isolated perfused rat kidney, cortisol was metabolized into cortisone and two polar metabolites, which have the same mobility on paper and on gas liquid chromatography than authentic 206-dihydrocortisol and 20g-dihydrocortisonerespectively, although no further identificationwas attempted, i.e. the separation between the 20a
and 208
epimers.
However, these results, on the nature of the main metabolites of# cortisol in kidney, are in good agreement with the previous data reported -in vitro in the rat. Using slices, minces, or homogenates, Mahesh 3 (1) found that H-cortisol was metabolized to cortisone, 20@-dihydrocortisol and 20@dihydrocortisone. However, the percent of conversion of cortisol to its metabolites differed widely according to the -in vitro method used. 'Inman, the formation of cortisone, 20@-dihydro was also shown by Jenkins (Z), by cortisol and 20f3-dihydrocortisone in vitro slices incubation. Only the @form was found. In dogs, 20 B-dihydrocortisol and 20@-dihydrocortisonehave also been identified -in vivo in urine and are the only cortisol metabolites found when the liver has been removed, indicating their extra-hepatic origin (7). Moreover, from the present study, it appeared that the rat kidney does not conjugate -in vivo cortisol and its metabolites. The absence of glucuroconjugationhas also been shown by Mahesh (1) and -in vivo for aldosterone, using the same isolated perfused rat kidney model (4). The isolated perfused rat kidney also permits a metabolic shortterm kinetic study . The kinetic rate constants can be easily determined and the following schema of the cortisol metabolism in kidney is proposed, from the data expressed in Fig. 6. Cortisol
b-D
20 reduced cortisol
Cortisone
20 reduced cortisone
As shown here, the 20-reduction of cortisone appears to be faster than
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633
that of cortisol. Finally, the renal handling of cortisol was studied with the use of this model. The urinary excretion of cortisol was found to be diuresis dependent as shown _in vivo in animals (8) and in man (9). As it is known that cortisol is submitted to a filtration-reabsorption process(lO), the influence of the diuresis on the urinary excretion of these steroids could be explained if one assumed that reabsorption decreases when diuresis increases. It should be noted that the relative concentration of 20-reduced cortisol was much higher in urine than in perfusate in the first periods of the perfusion. Since this compound is formed in the kidney and is recirculating in the perfusate, it is possible that the metabolite is more actively filtered and/or less reabsorbed than cortisol. This would explain why this metabolite was more rapidly excreted than cortisol. Alternatively, this compound may be synthetized in the kidney from cortisol, directly eliminated in urine and partially reabsorbed. Thus,a fraction of this metabolite may be directly excreted in urine without a recirculation in the perfusate. 20g-dihydrocortisoland 20B-dihydrocortisonehave been identified in human (11) and dog (12) urine. The origin of these compounds may he in the liver, although they are metabolized into S-cortol and S-cortolone (13). From our experiments, it is suggested that a fraction of these urinary compounds is formed in the kidney, then directly excreted in urine, escaping the hepatic metabolism. The isolated perfused rat kidney offers several advantages. However, the results obtained by the use of this model should he
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634
TIlEOXDb
compared with true -in vivo studies since steroid metabolism by the kidney in vivo depends on other factors, such as protein binding. The isolated perfused rat kidney may be a convenient model for studying the influence of such extra-renal factors on the renal handling of steroid hormones.
APPENDIX Consider the model :
k3
[
k'
'1
D
k2
C
The differential equations are as follows : dA - -(kl + k3)A zdB= dt
kB-kC 1
dD dt
kA3
and C = 1 - (A+B+D)
2
These equations integrate into (if A0 = 1, B
= Co = Do = 0): 0
A
B=
=
e
-0y
+
k3)t
k, + k?
(e -(kl+k3)t
_ e -k2t )
k2 - (k,+k3)
D=
k3
(1 - e -(kl+k3)t
kl+k3 ACKNOWLEDGMENTS This work has been supported by grants from Institut National de la Santd et de la Recherche Mddicale (Contrat no 744093-5).
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635
REFBRENCES
1. 2. 3. 4.
Mahesh, V.B. and Ulrich,F.,J. Biol. Chem. 235, 356 (1960) Jenkins, J.S., J. Endocrinol. 34, 151 (196r Bowman, R.H., J. Biol. Chem. 245, 1604 (1970) Nakane, H., Nakan6, Y., Rgach, G., Corvol, P. and MBnard,J., Am. J. Physiol., in press. 5. Vinson, G.P. and Whitehouse, B.J., In Advances in Steroid Bioche? mistry and Pharmacology, i, 163, Academic Press, New York and London (1970). 6. Nicholas, T.E., and Kim, P.A., Steroids, 25, 387 (1975) 7. Gold, N.I., J. Biol. Chem. 236, 1930 (196n 8. Kalant, N., Am. J. Physiol. 132, 503 (1955) 9. Hatfield, C.B., and Schuster,., J. Endocrinol. 2, 262 (1959) 10. Beisel, W.R., Di Raimondo, V.C. and Forsham, P.H., Ann. Int. Med. 2, 641 (1964) 11. Holness, N.J., Lunnon, J.B., and Gray, C.H., J. Endocrinol. 14, 136 (1956) 12. Gold, N.I., J. Biol. Chem. 236, 1924 (1961) 13. Bradlow, H.L., Fukushima, D.K., Zumoff, B., Hellman, L. and Gallagher, T.F., J. Clin. Endocr. Metab. 22, 748 (1962).