Effect of uremia on aldosterone metabolism in the isolated perfused rat liver

Effect of Uremia

on Aldosterone

Metabolism

in the Isolated

Perfused

Rat Liver

Martin Egfjord, Henrik Daugaard, and Klaus Olgaard The effect of uremia on hepatic metabolism of aldosterone was studied in the isolated perfused liver of female Wistar rats. Uremia was induced by five-sixths’ partial nephrectomy 4 weeks before experiments. Isolated livers of normal and uremic rats were perfused at a constant flow rate with a hemoglobin-free medium, to which 4-14C-o-aldosterone was added at 3 nmol/L. Aldosterone was analyzed by radioimmunoassay (RIA) and 4-14C-o-aldosterone radiometabolites in perfusate and bile were assayed by high-performance liquid chromatography (HPLC). Uremic rats had a 10% lower body weight (P c .Ol) and increased plasma urea, creatinine, and parathyroid hormone (PTH) levels (258%, 200%. and 208%. respectively; P c .Ol-.OOl). Blood pressure and plasma K+, Na+, and aldosterone levels were similar. Plasma renin activity was suppressed by 68% in uremic rats (P < ,001). Liver wet weight and hepatic function were similar in livers of both groups of rats. Hepatic elimination of aldosterone was compatible with a first-order kinetics. Hepatic clearance of aldosterone per liver and per gram liver was similar; however, when expressed per 100 g rat body weight, a 21% higher value was observed in uremic rats (11.8 f 1.8 mL/min) compared with normal rats (9.6 f 1.5 mL/min, P < .Ol). Polar aldosterone radiometabolites accumulated in the petfusate to approximately 40% of the initial 1% added at 15 minutes, and were eliminated in bile at a similar rate in both groups. No qualitative difference was found in the pattern of radiometabolites of aldosterone in perfusate and bile. Thus, despite a relatively increased capacity of hepatic aldosterone metabolism, chronic uremia in the rat did not specifically affect hepatic metabolism of aldosterone. Copyright 0 1993 by W.B. Saunders Company

A

LDOSTERONE IS THE MOST potent secreted corticosteroid of physiological importance for renal regulation of sodium and potassium excretion.* In uremic patients, great differences have been reported in plasma levels of aldosterone, which to a large extent correlated to variations in the stimulating factors on the adrenal zona glomerulosa.2-6 However, less attention has been directed toward the regulation of elimination of the hormone from circulation, and the metabolic clearance rate of aldosterone in chronic renal failure has been only sparsely examined. Plasma clearance rate of aldosterone was increased in nonnephrectomized patients with severe chronic renal disease of different etiology7,s and similar findings were reported in some anephric patients on maintenance hemoin plasma clearance of dialysis,2%9-11 while no changes aldosterone were found in other anephric patients.2~1’i~*2In chronic renal failure, hepatic metabolism of aldosterone might be stimulated by a high hepatic plasma flow.” However, we have previously found a higher hepatic clearance rate of prednisone in isolated livers of uremic rats than

From Medical Department P, Division of Nephrology, and the Department of E.xperimental Pathology, Rigshospitalet, University of Copenhagen, Denmark. Submitted February 5, 1992; accepted April 29, 1992. Supported by grants from the Alfred Benzons Foundation, the Danish Medical Research Council, the Dagmar Marshalls Foundation, the NOVOs Foundation, the Helen and Ejnar Bjamows Foundation, the Elin Hartehus Foundation, the Nordisk Insulin Foundation, the Dr August Petersen di Dr Thorkil Petersen Foundation, the Karla Marie Jorgensen Foundation, the King Christian Xs Foundation, the Foundation of IS70, the Foundation for Storkabenhavn, Fzraeme and Granland, the Soren A. Andersen Foundation, and the direktar Jacob Madsen and hustru Olga Madsen Foundation. Presented in pan at the 11th International Congress of Nephrology, Tokyo, Japan, July 15-20, 1990. Address reprint requests to Martin Egfiord, MD, Medical Department P 2131, Division of Nephrology, Rigshospitalet, 9 Blegdamsvej, 2100 Copenhagen, Denmark. Copyright 0 I993 by W.B. Saunders Company 0026-049519314204-0012$03.00/0 470

in isolated livers of normal rats when perfused with a constant flow rate.13.i4 The present study was therefore performed to examine the influence of chronic uremia under fixed experimental conditions on the metabolism of 4-i4C-D-aldosterone in the isolated perfused rat liver. MATERIALS

AND METHODS

Chemicals 4-i4C-D-Aldosterone (57 mCi/mmol) and 1,2-3H-3&B-tetrahydroaldosterone (58.6 Ci/mmol) were obtained from New England Nuclear, Boston, MA. Aldosterone-3-(O-carboxymethyl)oximino(2-*251-iodohistamine) ( - 2,000 Ci/mmol) was obtained from Radiochemical Center, Amersham, England. D-Aldosterone, 3a,58-, 3p,SB-, and 3B,Sa-tetrahydroaldosterone (THA), Su-dihydroaldosterone, I.+-lactic acid, D+-galactose, and bovine serum albumin fraction V (dialyzed) were obtained from Sigma, St Louis. MO. Aldosterone-I&glucuronide and llB, 18,18,20P-diepoxy-SB-pregnan-3o-ol were kindly supplied by professor D.N. Kirk, The Steroid Reference Collection, Queen Mary College, University of London. Aldosterone rabbit antiserum (SB-Aldo-H) was obtained from Commissariat a L’energie Atomique, Gif-Sur-Yvette, France. Steroid ‘251-diluent and precipitating reagent were obtained from ICN Biomedicals (RSL, Immuchem & Micromedic, Carson, CA). 8-Glucuronidase/sulfatase SHP (Helix pomatia juice) was obtained from Reactifs IBF, Villeneuve-La-Garenne, France. Scintillation liquid Ultima-Gold R was obtained from Packard Canberra. Groningen, The Netherlands. The organic solvents used for highperformance liquid chromatography (HPLC), methanol, ethanol, and 2-propanol, were all obtained from Li-Chrosolv R, Merck, Germany. In glucose, lactate, and galactose assays, enzymes from Boehringer. Mannheim, Germany, were used. All other chemicals were of analytical grade.

Analytical Procedures Oxygen tension (Pox) and glucose, lactate, and galactose concentrations in the perfusate were measured as previously described.13J5 Aldosterone concentrations were measured after organic extraction with ethyl acetateihexane (3:2voI/vol) by a specific radioimmunoassay (RIA), as previously described.i6~i7 Detection limit of the assay was 10 pg/mL; the within-assay coefficient of variation was

Metabolism,

Vol42, No 4 (April), 1993: pp 470-476

HEPATIC ALDOSTERONE

METABOLISM

471

IN UREMIC RATS

9.4% at 125 pg/mL and 10.4% at 1,000 pg/mL, and the betweenassay coefficient of variation was 10.8%. The recovery of aldosterone added (0 to 1,000 pg/mL) to a perfusate blank was 91% (r = .98). The cross-reactivity of the assay was as follows: aldosterone-18-glucuronide, 0.03%; 118,18,18,20B-diepoxy-5l3-pregnan-3o01,0.02%; So-dihydroaldosterone, 18%; 3a,SP-THA, < 10%; 38,58THA, 12%; 3B,Sa-THA, 15%; cortisol, dihydrocortisol, and tetrahydrocortisol, < 0.01%; corticosterone, < 0.02%; deoxycorticosterone acetate, 0.02%; and 18-hydroxycorticosterone, 0.03%. Total t4C radioactivity (DPM) was measured as follows: in duplicate, 1 mL perfusate or 50-ILL bile samples were added to 4 mL scintillation liquid and counted in a liquid scintillation counter (Tri-Carb 2200 CA, Packard Instruments, Downers Grove, IL). HPLC of 4-14C-o-aldosterone and its metabolites in perfusate and bile was performed after extraction on Sep-Pak Cl8 cartridges (Millipore, Waters Associates, Millford, MA) and elution with methanol15 on a reverse-phase Nucleosil5-urn C8 column (50 x 46mm + 250 x 46-mm, Macherey-Nagel, Duren, Germany), as previously described.i6,t7 The mobile phase was propanoliwater (25:75 vol/vol) at 40°C at a flow rate of 0.7 mL/min. Fractions were collected initially every 0.5 minutes and then every 2 minutes from 30 to 60 minutes of run; 4 mL scintillation liquid was added and counted in a Packard Tri-Carb 2200 CA liquid scintillation counter. The recovery of t4C radioactivity after Sep-Pak extraction was approximately 70%, and after HPLC it was almost 100%. To hydrolyze eventual steroid conjugates, parallel samples extracted on Sep-Pak were treated with @glucuronidase/sulfatase (Helix pomatia juice) before performing chromatographyt6 at pH 4.5 and 37°C in darkness for 70 hours; samples were then reextracted on Sep-Pak Cl8 cartridges and chromatographed as previously described. p-Creatinine,p-urea,p-Na+, andp-K+ were measured by routine methods on an autoanalyzer. Plasma renin activity was measured with a RIANEN 1251angiotensin I RIA kit obtained from New England Nuclear, as previously described.i6 Plasma parathyroid hormone (PTH) was measured with an N-terminal RIA, INS-PTH, obtained from the Nichols Institute, San Juan Capistrano, CA, as previously described.18

Experimental Chronic Uremia Female Wistar rats weighing approximately 180 g were obtained from Mollegaards Breeding Center, Ejby, Denmark. Experimental chronic uremia was induced by five-sixths’ nephrectomy as described previously.i3,t4 Under pentobarbital anesthesia (6 mg/lOO g rat body weight), approximately two thirds of the left kidney and the entire right kidney were removed, with careful preservation of both adrenal glands, in a one-step operation. Both partially nephrectomized and control rats were kept in a room with a constant temperature (22°C) and a fixed artificial light cycle (light 7:00 AM to 6:00 PM), and were fed a standard rat chow containing 0.2% sodium and 1% potassium (Altromin International rat/ mouse maintenance 1320, Lage, Germany) and allowed water ad libitum. All experiments were performed 3 to 4 weeks later. Rats were fasted for 24 hours before the experiments, but were allowed free access to drinking water. The degree of uremia was measured at the time of liver perfusion by plasma urea, creatinine, and PTH levels. Arterial blood pressure, plasma renin activity, and aldosterone and PTH levels were measured in pentobarbital-anesthetized rats (6 mg/lOO g rat body weight) after abdominal incision. The abdominal aorta was punctured by a 21-gauge x 1.5-inch cannula, and direct measurements of the systolic blood pressure were obtained as previously described.16 The animals were then exsangui-

nated; the blood was immediately chilled in tubes containing EDTA and separated at 4°C. Plasma samples were stored at -20°C until analysis. Isolated Perfused Rat Liver The perfusion medium was a hemoglobin-free isotonic modified Krebs-Henseleit bicarbonate solution containing sodium chloride 110 mmol/L, potassium chloride 3.1 mmol/L, potassium dihydrogen phosphate 2.0 mmol/L, magnesium sulfate 0.8 mmol/L, calcium chloride 2.4 mmol/L, and sodium hydrogen carbonate 25 mmol/L. Bovine serum albumin fraction V was added at 50 g/L, L+-lactic acid at 10 mmol/L, and o+-galactose at 3 mmol/L. The perfusate was prefiltered and gassed with 95% 02 and 5% COz. The perfusion apparatus and the operative technique were as previously described13 from our laboratory. In pentobarbital anesthesia, the common bile duct was cannulated with PE 50 tubing for bile collection; the portal vein and the inferior caval vein were cannulated with 14-gauge catheters. Immediately before liver experiments, blood (1 mL) was sampled for measurements of plasma creatinine, urea, Na+, and K+ concentrations. Isolated livers were perfused in a porto-caval direction at 37°C in a recirculating mode with a constant perfusion rate of 35 mL/min. The viability of perfused livers was assessed by their macroscopic appearance, portal vein pressure, bile flow, oxygen consumption, rate of gluconeogenesis from 10 mmol/L lactate, and rate of galactose elimination from perfusate. Perfusion Experiments After an equilibration period of 30 minutes, the perfusate was exchanged with fresh medium containing 4-14C-D-aldosterone at a concentration of 3 nmol/L (_ 1,170 pg/mL) and recirculation with a total perfusate volume of 150 mL was established.t6 Samples of 7 mL for measurements of aldosterone, 14C radioactivity, glucose, lactate, and galactose concentrations were obtained from the reservoir at 0, 5, 10, 15,30, 60, and 90 minutes, and the remaining perfusate was collected. Samples for POZ determinations (1 mL) were drawn simultaneously from catheters in the portal and caval veins at 15 and 60 minutes. Bile was collected continuously, and 14C radioactivity was examined. Samples for measurements of aldosterone and 14C radioactivity and HPLC were kept at -20°C until analysis. Calculations Hepatic oxygen consumption, the average rate of gluconeogenesis, and the average elimination rate of lactate and galactose were calculated as previously described.16 Perfusate aldosterone concentrations were corrected for the influence of sampling from the reservoir.t4 Hepatic clearance of aldosterone was then determined as previously described.16 A perfusate concentration-time curve of the radiometabolites of 4-14C-o-aldosterone was achieved by subtraction of the total 14C and the 4-i4C-o-aldosterone radioactivity concentration-time curves in each experiment. l6 The biliary clearance of 14Cradiometabolites (Bi Cli4c ,& was estimated by the equation, Bi Clic me, = average bile flow x concentration of 14C radiometabolites in bile/median concentration of 14Cradiometabolites in perfusate.t9 Statistical Methods Results are means f SD unless otherwise indicated. Parametric coefficients of correlation and simple linear regression analysis were calculated. Student’s t test was used to estimate the significance of differences between mean values; P < .05 was considered statistically significant.

472

EGFJORD, DAUGAARD, AND OLGAARD

RESULTS

Table 2. Functional Parameters of Isolated Perfused Livers of Normal

Uremic rats had a reduced weight gain, resulting in 10% lower body weight at the time of experimentation (P < .Ol). Renal function, when assessed by plasma urea and creatinine levels, was reduced in the partially nephrectomized rats (P < .Ol-.OOl). This was further supported by increased levels of PTH in uremic rats (P < .Ol). In fasted rats, similar levels of plasma potassium and sodium and no difference in the systolic arterial blood pressure were observed. Plasma renin activity was reduced in uremic rats (P < .OOl). However, similar levels of plasma aldosterone were found (Table 1). As for liver wet weight and the functional parameters of isolated perfused livers, portal vein pressure, bile flow, oxygen consumption, glucose formation, and galactose elimination per liver and per gram liver wet weight were similar in both groups of rats. Only hepatic formation of glucose, when expressed per 100 g rat body weight, was 19% higher (P < .05) in livers of uremic rats (Table 2). The disappearance rate of aldosterone, added to the perfusate at 3 nmol/L, was similar in isolated livers of normal and uremic rats. In livers of both normal and uremic rats, a positive linear regression was found between the mean perfusate aldosterone concentration and the average hepatic elimination rate of aldosterone per gram liver wet weight in each sampling interval (r = .97 and .95, respectively; P < .OOl). In normal and uremic rats, hepatic clearance of aldosterone per liver and per gram liver was similar (Table 3). However, when expressed per 100 g rat body weight, a 21% higher value was observed in uremic rats compared with normal rats (P < .Ol; Table 3). Total r4C radioactivity in perfusate decreased similarly in livers of normal and uremic rats. The calculated perfusate 14Caldosterone radiometabolite concentration increased similarly within the first 10 to 15 minutes to approximately 40% of the initial i4C added in livers of both groups of rats, and did not differ during the experimental period (Fig 1). No difference in total biliary excretion of i4C radioactivity and in biliary clearance of 14C-aldosterone metabolites during the 90 minutes of experiments was observed (Table 3). In all liver perfusion experiments, a positive correlation was found between total biliary excretion of i4C and bile flow (r = .75, P < .OOl; n = 20). A negative correlation was

and Uremic Rats Normal Rat

Liver wet weight(g) Portal vein pressure maximal (cm water) Average bile flow per liver (mL/h)

Uremic

(n = 11)

(n = 9)

6.5 + 0.6

6.7 + 0.9

1oc

1

0.23 f 0.07

11 k 1 0.27 k 0.15

Oxygen consumption (pm01 O*/min) Per liver

14.7 ‘c 2.4

15.9 k 3.1

Per g liver

2.28 f 0.43

2.38 2 0.40

Per 100 g rat

7.26 z 1.47

6.67 -t 1.60

Glucose formation (pmol/min) 4.0 5 0.7

4.4 t 0.9

Par g liver

0.63 t 0.12

0.65 + 0.13

Per 100 g rat

1.98 -t 0.37

2.36 k 0.37*

2.1 5 0.7

1.6 it 0.6

Per liver

Delta lactate/delta glucose Galactose elimination (kmol/min) Per liver

0.98 -+ 0.27

0.92 2 0.25

Per g liver

0.15 + 0.04

0.14 f 0.04

Per 100 g rat

0.48 2 0.15

0.50 ? 0.14

NOTE. Results are means k SD. *Different from normal rats (P < .O!i).

observed between total biliary excretion of ‘Y and perfusate *4C-aldosterone radiometabolite concentration at 90 minutes (r = -.96, P < .OOl;n = 20). However, no correlation between hepatic clearance of aldosterone and total biliary excretion of i4C was observed (r = - .06, NS; n = 20). Characterization of Metabolites

The reverse-phase HPLC method separated aldosterone from its more polar metabolites, 17-iso-aldosterone, and the reduced metabolites, Sa-dihydroaldosterone and 3u,SpTHA. The stereoisomeric forms of THA could not be clearly separated.i6J7 The relative retention times when compared with D-aldosterone were as follows: aldosterone18-glucuronide, 0.3; 17-iso-aldosterone, 0.9; Sa-dihydroaldosterone, 1.3; and 3a,S@THA, 1.8. The i4C pattern in perfusate samples obtained from the two groups of livers in the present study was analyzed after 15 minutes of perfusion. Approximately 1 mL of the perfusate samples from all experiments in each group were Table 3. Metabolism of 4-W-o-Aldosterone

in Isolated Perfused

Livers of Normal and Uremic Rats Table 1. Renal Function, Systolic Blood Pressure, Plasma Renin

Rat

Activity, and Plasma Aldosterone Level in Normal and Uremic Rats Rat

NOUW3l

203 k 10 (11)

183 k 17 (9)”

Plasma urea (mmol/L)

7.1 k 1.4 (11)

18.3 2 8.3 (9)t

concentration (pg/mL)

Per liver

Plasma K+ (mmol/L)

3.1 + 0.4(11)

3.1 + 0.4 (9)

Plasma Nat (mmol/L)

134 + 14 (11)

132 ? 13 (9)

Per g liver Per 100 g rat

25 + 17 (16)*

Blood pressure in anesthetized rats (mm Hg) Plasma renin activity (ng/mL/h) Plasma aldosterone (pg/mL)

1,160 2 140

1,178 + 76

(mL/min)

106 f 48 (9)*

12 + 4 (14)

Ill= 9)

Hepatic clearance of aldosterone

14(11)

Plasma PTH 1-34 (pg/mL)

532

Uremic

Initial aldosterone perfusate Uremic

Rat weight(g) Plasma creatinine (Fmol/L)

Normal In = 11)

Mary

19.4 + 3.1 3.0 + 0.4 9.6 + 1.5

21.3 2 4.3 3.3 2 0.8 11.62 1.8*

clearance of r4C-aldosterone

metabolites (mL/min) 100 k

15 (16) 12 k 7 (16)t

Per liver

3.2 -c 2.1

2.7 2 2.3

37 k 16 (14)

0.5 + 0.4

0.4 2 0.4

773 t 366 (14)

608 -c 536 (16)

Per g liver Per 100 g rat

1.6?

1.5 + 1.2

94 + 10 (14)

NOTE. Results are means c SD: number of rats is in parentheses.

NOTE. Results are means 2 SD.

‘P < .Ol, tf

*Different from normal rats (P < .Ol).

< ,001, different from normal rats.

1.1

HEPATIC ALDOSTERONE

METABOLISM

473

IN UREMIC RATS

PERFUSATE

pooled. At 15 minutes, average perfusate aldosterone levels decreased to 8% of the initial value and average calculated perfusate 14C-radiometabolite levels were 40% (Fig 1). Correspondingly, the 14Cpeaks coeluating with aldosterone were relatively small. Most of the metabolites consisted of several peaks more polar than that of aldosterone. A minor peak less polar than that of THA was also observed in both groups (Fig 2). *4C-radiometabolite patterns in perfusate and bile after 90 minutes of perfusion were examined in two livers in each group of rats in the present study. Perfusate aldosterone levels were below 1% of the initial value. At 90 minutes, only the polar aldosterone metabolites with a major peak corresponding to the void volume were detected in perfusate of both groups. After hydrolysis of the samples with glucuronidase/sulfatase, the polar peaks remained unchanged in both groups of rats (Fig 3). m Y

15 minutes

A

x

-a-

14c

-

3X-TiiA

-

ALL)0

DPY

8

3 B

0

x

:-

0

r:

8

8

fraction

k::::::::::::::::;

B ;

50--

Fig 2. HPLC separation of 4-‘4C-D-aldosterone metabolites in perfusate obtained after 15 minutes of perfusion from isolated perfused livers of normal (A) end uremic (6) female rats in this study. Retention times of D-aldosterone [ALDO) and 1,2-“H-3o,5B-THA (3HTHA) are marked.

;f F

The biliary pattern of aldosterone radiometabolites is also shown in Fig 3. Only the most polar i4C metabolites of aldosterone were excreted in bile; after enzymatic hydrolysis, the i4C metabolites remained more polar than aldosterone. Thus no qualitative differences were found in the chromatograms between normal and uremic rats.

25 --

g d 0

t t

DISCUSSION

Minutes Fig 1. (A) 4-r4C-D-aldosterone perfusate concentration (%) and (8) calculated perfusate 4JF-o-rldosterone metabolites concentration (X of initial total 1% added) Y time (mean -C SEM) in isolated perfused livers obtained from normal and uremic female Wistar rats. Data are from isolated livers obtained from 11 normal (0) and nine uremic rats (X) all perfused with an initial aldosterone concentration of 3 nmol/L. Control perfusions without a liver in the circuit were performed to examine for possible nonspecific adhesion and breakdown of 4-“C-Daldosterone in the apparatus (N = 4 lo]).

In isolated rat liver, aldosterone was extensively metabolized within the first 30 minutes of perfusion and converted to metabolites more polar than aldosterone. Aldosterone metabolites were both released back into the circulation from the liver and eliminated in bile. Enzymatic hydrolysis did not affect polar aldosterone metabolites in both perfusate and bile, indicating an unconjugated nature of these aldosterone metabolites. This was in accordance with our previous findings in the isolated perfused liver of normal

474

EGFJORD, DAUGAARD,

AND OLGAARD

fraction

fraction

BILE

A

800

100 ZOO 0 3 fraction

8

0

s fraction

%

Fig 3. HPLC separation of 4-W-p-aldosterone metabolites in perfusate (upper panel) and bile (lower panel) obtained after 90 minutes of perfusion from isolated perfused Ihrers of normal (A and C) and uremic (6 and D) female rats. To hydrolyze eventual steroid cor@qates, half of the samples were treated with f3ghicuronidaN sutfatase before the HPLC run (C and 0).

HEPATIC ALDOSTERONE

METABOLISM

475

IN UREMIC RATS

female rats.16 A precise characterization of these hepatic polar metabolites of aldosterone requires further study. At the time of liver perfusions, the partially nephrectomized rats exhibited a moderate degree of chronic renal failure, as measured by increased levels of plasma creatinine and urea (P < .Ol-.OOl), comparable with renal function data obtained by others using this experimental mode1.20-2rThe presence of uremia in partially nephrectomized rats was further supported by the observed reduced gain in rat body weight and by elevated levels of PTH (P < .Ol). Furthermore, uremic rats in the present study exhibited reduced plasma renin activities when compared with normal rats (P < .OOl), which probably represented an adaptation to a reduced ability of renal sodium excretion in uremic rats at normal arterial blood pressure.23,24 Fasting plasma potassium and sodium levels were unaffected in partially nephrectomized rats, in agreement with an adaptation of the persisting nephrons resulting in an increased fractional excretion of sodium and potassium in renal failure.24,25 Plasma aldosterone levels in normal female Wistar rats on a standard sodium diet in the present study were in accordance with previously reported levels in sodium-unrestricted female Wistar rats. 26The unchanged plasma levels of aldosterone, despite reduced plasma renin activity in uremic rats of the present study, could indicate that the observed normokalemia in chronic renal failure was maintained by an increased aldosterone secretion, as reported in other studies.27-29 The functional parameters of isolated perfused livers and total hepatic clearance of aldosterone were not affected by the preexisting uremia, and no qualitative changes in the pattern of hepatic aldosterone metabolites were observed. However, hepatic clearance of aldosterone, when expressed per 100 g rat body weight, was 21% higher in uremic rats compared with normal rats (P < .Ol). This relatively increased capacity of hepatic metabolism of aldosterone was in parallel with our previous findings of a 36% higher clearance rate of prednisone in isolated perfused livers of uremic rats when compared with livers of normal rats.16J7 However, uremia resulted in an absolute increase in hepatic clearance of prednisone, while total hepatic clearance

of aldosterone

was not affected.

The

value of hepatic clearance of aldosterone (19.4 2 3.1 mL/ min/liver [N = 111) was 80% higher (P < .OOl) than that previously reported for prednisone (10.8 ? 2.4 mL/min/ liver [N = 121)measured at a circulating prednisone concentration of 600 ng/mL in normal Wistar rats of a similar age and sex.14 The different magnitude of these hepatic clearance rates was probably not explained by differences in circulating levels of the two steroids; when isolated livers of normal female Wistar rats were perfused with aldosterone at 60 to 120 ng/mL, hepatic clearance of aldosterone was still unchanged (19.0 r 5.4 mL/min, N = 5; unpublished observations) and significantly higher (P < .OS) than hepatic clearance of prednisone. Thus, these studies indicate that different hepatic corticosteroid-metabolizing enzymes are involved in the metabolism of aldosterone and prednisone, which could explain the different relative sensitivity to the stimulatory effect of uremia. Thus, the use of the isolated perfused liver made it possible to assess the effects of preexisting uremia on the hepatic metabolism of aldosterone under fixed experimental conditions at a constant flow rate, without the influence of altered secretion rate and volume of distribution of the hormone. In the isolated liver of female rats, aldosterone at physiological concentrations was rapidly converted to more polar metabolites, which supplied evidence for the importance of the liver in the maintenance of circulating levels of aldosterone. Introduction of moderate uremia resulted in a relatively higher capacity of hepatic metabolism of aldosterone when expressed per gram body weight, which together with the suppressed plasma renin activity might contribute to a down-regulation of plasma aldosterone and renal reabsorption of sodium. However, no specific effect of chronic uremia on hepatic metabolism of aldosterone was found. ACKNOWLEDGMENT

Professor D.N. Kirk is thanked for generous gifts from The Steroid Reference Collection, Queen Mary College, University of London. We also wish to thank Kirsten Bang, Liselotte Damsgaard, Betty Fischer, and Vibeke Pless for skillful technical assistance.

REFERENCES 1. Simpson SA, Tait JF: Recent progress in methods of isola-

tion, chemistry and physiology of aldosterone. Recent Prog Horm Res 11:183-219, 1955 2. Williams GH, Bailey GL, Hampers CL, et al: Studies on the metabolism of aldosterone in chronic renal failure and anephric man. Kidney Int 4280-288.1973 3. Weidmann P, Maxwell MH, Lupu AN: Plasma aldosterone in terminal renal failure. Ann Intern Med 78:13-18, 1973 4. Weidmann P, Maxwell MH, deLima J, et al: Control of aldosterone responsiveness in terminal renal failure. Kidney Int 7:351-359,1975 5. Henrich WL, Katz FH, Molinoff PB, et al: Competitive effects of hypokalemia and volume depletion on plasma renin activity, aldosterone and catecholamine concentrations in hemodialysis patients. Kidney Int 12:279-284,1977 6. Olgaard K: Plasma aldosterone regulation in anephric and

non-nephrectomized patients on regular hemodialysis. A survey. Dan Med Bull 27:22-35,198O 7. Lommer D, Dusterdieck G, Jahnecke J, et al: Sekretion, Plasma-konzentration, Verteilung, Stofiechsel und Ausscheidung von Aldosteron bei Gesunden und Kranken. Klin Wochenschr 46:741-751,1968 8. Vecsei P, Dusterdieck G, Jahnecke J, et al: Secretion and turnover of aldosterone in various pathological states. Clin Sci 36:241-256,1969 9. Balikian HM, Brodie AH, Dale SL, et al: Effect of posture on the metabolic clearance rate, plasma concentration and blood production rate of aldosterone in man. J Clin EndocrinoI 28.16301640,1968 10. Weidmann P, Horton R, Maxwell MH, et al: Dynamic studies of aldosterone in anephric man. Kidney Int 4:289-298,1973 11. Corvol P, Bertagna X, Bedrossian J: Increased steroid

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metabolic clearance rate in anephric patients. Acta Endocrinol 75:7X-762, 1974 12. Read VH, McCaa CS, Bower JD, et al: Effect of hemodialysis on the metabolic clearance rate, plasma concentration and blood production rate of aldosterone in anephric man. J Clin Endocrinol Metab 361773-778, 1973 13. Egfjord M, Langhoff E, Daugaard H, et al: Increased clearance rate of prednisone in the isolated perfused liver of uremic rats. Nephron 45:53-58, 1987 14. Egfjord M, Daugaard H, Olgaard K: Clearance of prednisone in isolated perfused livers of normal and uremic rats: Effects of acute and chronic cyclosporin-A administration. Drug Metab Dispos 18:231-23?,1990 15. Bergmeyer HU. Bergmeyer J, Grass1 M: Methods of Enzymatic Analysis, vol 6 (ed 3). Weinheim, Germany, Verlag Chemie. 1984, pp 163-1?8,281-296,582-588 16. Egfjord M, Olgaard K: Aldosterone metabolism in the isolated perfused liver of female and male rats. Endocrinology 125:3011-3021.1989 17. Egfjord M, Daugaard H, Olgaard K: Aldosterone metabolism in combined isolated perfused liver and kidney. Am J Physiol 260:F536-F548,1991 18. Staun M, Egfjord M, Daugaard H, et al: Renal and intestinal calcium-binding proteins in normal and uraemic rats. Stand J Clin Lab Invest 51:111-118,199l 19. Rowland M, Tozer TN: Clinical Pharmacokinetics: Concepts and Applications (ed 2). Philadelphia, PA, Lea & Febiger, 1989, p 160

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20. Kaufman JM, DiMeola HJ, Siegel NJ, et al: Compensatory adaptation of structure and function following progressive renal ablation. Kidney Int 6:10-l?, 1974 21. Rabkin R, Unterhalter SA, Duckworth WC: Effect of prolonged uremia on insulin metabolism by isolated liver and muscle. Kidney Int 16:433-439, 1979 22. Ormrod D, Miller T: Experimental uremia: Description of a model producing varying degrees of stable uremia. Nephron 26:249-254, 1980 23. Bourgoignie JJ, Kaplan M. Gavellas G, et al: Sodium homeostasis in dogs with chronic renal insufficiency. Kidney Int 21:820-826,1982 24. Bricker NS: Sodium homeostasis in chronic renal disease. Kidney Int 21:886-897,1982 25. Simon ES, Martin D, Trigg D. et al: Contribution of the distal tubule to potassium excretion in experimental glomerulonephritis. Kidney Int 34:53-59, 1988 26. Braley LM, Menachery AI, Williams GH: Specificity of the alteration in aldosterone biosynthesis in the spontaneously hypertensive rat. Endocrinology 112:562-566,1983 27. Berl T, Katz FH, Henrich WL. et al: Role of aldosterone in the control of sodium excretion in patients with advanced chronic renal failure. Kidney Int 14:228-235. 1978 28. Sugarman A. Brown RS: The role of aldosterone in potassium tolerance: Studies in anephric humans. Kidney Int 34:39?403. 1988 29. Young DB: Qualitative analysis of aldosterone’s role in potassium regulation. Am J Physiol255:F811-F822, 1988