Institut für Pharmakologie, Medizinische Hochschule Hannover, Federal Republic of
Germany
URINARY ALDOSTERONE EXCRETION RATE AND PLASMA ALDOSTERONE CONCENTRATION IN THE RAT:
EFFECT OF ACTH, DOC, FUROSEMIDE AND OF CHANGES IN SODIUM BALANCE
By Mathias Hilfenhaus ABSTRACT In rats kept under standard conditions urinary aldosterone excretion rate (UAER) and plasma aldosterone concentration (PAC) showed a circadian rhythm with peak values at the beginning of the dark period and lowest values of the beginning of the light period. In rats treated with a single dose of DOC (2 mg/kg sc) or given saline as drinking water UAER and PAC decreased to very low levels. Restriction of dietary sodium for 5 weeks increased UAER and PAC 60-fold and 100-fold, respectively. Treatment with ACTH (250 or 500 \g=m\g/kgiv) stimulated UAER 8 h following injection and PAC 1 h after injection. In rats treated with a single dose of furosemide (25 mg/kg sc) PAC increased to maximal values within 90 min and reached control values within 24 h. Twenty-four hours\x=req-\ UAER also increased after furosemide treatment. UAER paralleled PAC under all experimental conditions tested. Therefore, our results suggest that UAER is a reliable index of the activity of the renin-angiotensin-aldosterone system in rats under conditions of low and high aldosterone secretion. Measurement of UAER is a useful tool for studies on aldosterone levels in rats over a long period of time.
Studies on aldosterone levels in rats have been performed by radioimmunologic determinations of plasma aldosterone concentration (PAC) (Campbell et al. 1974, 1975; Coleman et al. 1974; Kubo et al. 1974; Hilfenhaus 1974, 1975; Dietz et al. 1975). However, serial determinations of PAC in individual rats can not be obtained, since rats have to be sacrificed due to the considerable amount of plasma needed. 134
In contrast to plasma levels, urinary excretion rates of aldosterone (UAER) often used in man for diagnostic or research purposes are easy to measure over a long period of time even in small laboratory animals like the rat. Only a few reports have dealt with this parameter in the rat (Paroli 8c Melchiorri 1961; Loyke 1964; Schwartz et al. 1964) and studies on the biologic reliability of UAER as a measure of the activity of the renin-angiotensin-aldosterone system in the rat are lacking. In the present study UAER was compared to PAC in rats with different aldosterone levels induced by changes in sodium balance or by treatment with DOC, ACTH or the diuretic furosemide.
-
-
MATERIALS AND METHODS Male Sprague-Dawley rats (200-250 g) were kept on a standard diet (Na+ 96 mmol/kg, K+ 143 mmol/kg; Altromin®, Lage, Germany) and tap water ad libitum. In the ex¬ periments with sodium restriction rats received a sodium-poor diet (Na+ 4.4 mmol/kg, K+ 60 mmol/kg; Altromin®) and demineralized water ad libitum. Maintenance of the animals and sampling of plasma and urine were as previously described (Hilfenhaus
1976(3,(5). [1,2,6,7-3H] aldosterone (103 Ci/mmol) and [1,2,6,7-SH] corticosterone (96 Ci/mmol) were purchased from the Radiochemical Centre, Amersham. D-aldosterone and ACTH (/S'-24-ACTH, Synachten®) were kindly supplied by Ciba-Geigy. Deoxycorticosterone oenanthate (Cortiron-Depot®) was a gift by Schering and furosemide (Lasix®) by
Hoechst. Determination of urinary free aldosterone was performed as described for plasma aldosterone (Hilfenhaus 1976a), i. e. by radioimmunoassay preceded by thin layer chromatography. Reliability criteria were checked for urinary aldosterone determina¬ tion and found to completely suffice under this condition. Acid hydrolysis of urine was omitted, since it only resulted in a less than 10% increase of the aldosterone values obtained without hydrolysis. Determination of plasma corticosterone concentra¬ tion by radioimmunoassay has been described previously (Hilfenhaus 1976a). Urinary sodium was quantitated by flame photometry with an internal lithium standard. Student's t-test was used to assess the significance of differences between means.
RESULTS
Rats kept in metabolic cages in our experiments did not differ from rats kept in usual macrolon cages in respect to their plasma corticosterone and plasma aldosterone concentrations. Comparison of PAC and UAER in rats showed a parallelism in time-course and extent for the whole 24 h-cycle (Fig. 1). Highest values of PAC and UAER were observed at the beginning of the dark period and the lowest values at the beginning of the light period. For both parameters the ampli¬ tudes of the circadian rhythm (calculated as differences between the lowest and highest values) were in the range of 1:3. 135
18o»
6°°
6°°
TIME OF DAY
Fig.
1.
aldosterone concentration (PAC) and urinary aldosterone rhythm excretion rate (UAER) in male Sprague-Dawley rats. For measurement of PAC groups of rats were sacrificed at 3 h-intervals. For measurement of UAER urine was sampled in 6 h-portions. Solid black abscissa indicates time of darkness. Values represent mean ± sem (N 6).
Circadian
plasma
of
=
D
D
DOC
75
O
*
4
i
o —
\h
1
iI
2
3
l
8°° 18°° 5. DAY
5
A
DAY OF TREATMENT
Fig.
il
2.
UAER and PAC in rats drinking saline or treated with DOC. NaCl: Rats received 0.9 °/o saline as drinking water for 5 days. DOC: Rats received a single dose of deoxycorticosterone oenanthate (2 mg/kg sc) on the 1st day of the experiment. PAC was measured in rats sacrificed at 08.00 or at 18.00 on the 5th day of the experiment. Values are means + sem (N 6). =
136
300-
200-
W §í 100
-aefíC 1
28
35
39 DAYS
-^-Hn
F
ï5 3 0.01. b) P < 0.01. c) P < 0.001
as
compared
to the
corresponding
Fig.
control.
4.
UAER, PAC and plasma corticosterone concentration in
rats treated with ACTH. dissolved in 0.5 ml of isotonic laevulose solution and injected into a tail vein at 09.00. Controls received 0.5 ml of vehicle only. Urine was collected for the following 8 h and blood was collected 1 h after treatment. Plasma concentrations of aldosterone and corticosterone were measured in the same animals. Values represent means ± sem (N 6).
/J1-24-ACTH
was
=
138
Fig.
5.
PAC and UAER in rats treated with a single dose of furosemide (25 mg/kg sc) (Fur) at the start of the experiment (09.00). For measurement of PAC groups of rats were sacrificed at the times indicated. For comparison the 24 h-course of PAC in control rats (Co) is given (see Fig. 1). For determination of UAER urine was collected for 24 h from rats treated with furosemide or ¡sotonic laevulose at 09.00. Values represent means + sem (N 6). Solid black abscissa indicates time of darkness. =
In rats kept on sodium-poor diet for 5 weeks (Fig. 3) urinary sodium ex¬ cretion declined below 1/200 of the controls within 3 days returning to control levels within 3 days after re-feeding the standard diet. UAER gradually in¬ creased to levels 60 times about controls during 3 weeks and remained that high for the following 2 weeks. After re-feeding of standard diet UAER rapidly decreased to nearly normal values within 3 days. PAC increased more than 100-fold after 2 weeks of sodium restriction (Table 1). Within 4 days of re-feeding the standard diet PAC reached control values, while UAER remained slightly, but significantly (P
Germany
URINARY ALDOSTERONE EXCRETION RATE AND PLASMA ALDOSTERONE CONCENTRATION IN THE RAT:
EFFECT OF ACTH, DOC, FUROSEMIDE AND OF CHANGES IN SODIUM BALANCE
By Mathias Hilfenhaus ABSTRACT In rats kept under standard conditions urinary aldosterone excretion rate (UAER) and plasma aldosterone concentration (PAC) showed a circadian rhythm with peak values at the beginning of the dark period and lowest values of the beginning of the light period. In rats treated with a single dose of DOC (2 mg/kg sc) or given saline as drinking water UAER and PAC decreased to very low levels. Restriction of dietary sodium for 5 weeks increased UAER and PAC 60-fold and 100-fold, respectively. Treatment with ACTH (250 or 500 \g=m\g/kgiv) stimulated UAER 8 h following injection and PAC 1 h after injection. In rats treated with a single dose of furosemide (25 mg/kg sc) PAC increased to maximal values within 90 min and reached control values within 24 h. Twenty-four hours\x=req-\ UAER also increased after furosemide treatment. UAER paralleled PAC under all experimental conditions tested. Therefore, our results suggest that UAER is a reliable index of the activity of the renin-angiotensin-aldosterone system in rats under conditions of low and high aldosterone secretion. Measurement of UAER is a useful tool for studies on aldosterone levels in rats over a long period of time.
Studies on aldosterone levels in rats have been performed by radioimmunologic determinations of plasma aldosterone concentration (PAC) (Campbell et al. 1974, 1975; Coleman et al. 1974; Kubo et al. 1974; Hilfenhaus 1974, 1975; Dietz et al. 1975). However, serial determinations of PAC in individual rats can not be obtained, since rats have to be sacrificed due to the considerable amount of plasma needed. 134
In contrast to plasma levels, urinary excretion rates of aldosterone (UAER) often used in man for diagnostic or research purposes are easy to measure over a long period of time even in small laboratory animals like the rat. Only a few reports have dealt with this parameter in the rat (Paroli 8c Melchiorri 1961; Loyke 1964; Schwartz et al. 1964) and studies on the biologic reliability of UAER as a measure of the activity of the renin-angiotensin-aldosterone system in the rat are lacking. In the present study UAER was compared to PAC in rats with different aldosterone levels induced by changes in sodium balance or by treatment with DOC, ACTH or the diuretic furosemide.
-
-
MATERIALS AND METHODS Male Sprague-Dawley rats (200-250 g) were kept on a standard diet (Na+ 96 mmol/kg, K+ 143 mmol/kg; Altromin®, Lage, Germany) and tap water ad libitum. In the ex¬ periments with sodium restriction rats received a sodium-poor diet (Na+ 4.4 mmol/kg, K+ 60 mmol/kg; Altromin®) and demineralized water ad libitum. Maintenance of the animals and sampling of plasma and urine were as previously described (Hilfenhaus
1976(3,(5). [1,2,6,7-3H] aldosterone (103 Ci/mmol) and [1,2,6,7-SH] corticosterone (96 Ci/mmol) were purchased from the Radiochemical Centre, Amersham. D-aldosterone and ACTH (/S'-24-ACTH, Synachten®) were kindly supplied by Ciba-Geigy. Deoxycorticosterone oenanthate (Cortiron-Depot®) was a gift by Schering and furosemide (Lasix®) by
Hoechst. Determination of urinary free aldosterone was performed as described for plasma aldosterone (Hilfenhaus 1976a), i. e. by radioimmunoassay preceded by thin layer chromatography. Reliability criteria were checked for urinary aldosterone determina¬ tion and found to completely suffice under this condition. Acid hydrolysis of urine was omitted, since it only resulted in a less than 10% increase of the aldosterone values obtained without hydrolysis. Determination of plasma corticosterone concentra¬ tion by radioimmunoassay has been described previously (Hilfenhaus 1976a). Urinary sodium was quantitated by flame photometry with an internal lithium standard. Student's t-test was used to assess the significance of differences between means.
RESULTS
Rats kept in metabolic cages in our experiments did not differ from rats kept in usual macrolon cages in respect to their plasma corticosterone and plasma aldosterone concentrations. Comparison of PAC and UAER in rats showed a parallelism in time-course and extent for the whole 24 h-cycle (Fig. 1). Highest values of PAC and UAER were observed at the beginning of the dark period and the lowest values at the beginning of the light period. For both parameters the ampli¬ tudes of the circadian rhythm (calculated as differences between the lowest and highest values) were in the range of 1:3. 135
18o»
6°°
6°°
TIME OF DAY
Fig.
1.
aldosterone concentration (PAC) and urinary aldosterone rhythm excretion rate (UAER) in male Sprague-Dawley rats. For measurement of PAC groups of rats were sacrificed at 3 h-intervals. For measurement of UAER urine was sampled in 6 h-portions. Solid black abscissa indicates time of darkness. Values represent mean ± sem (N 6).
Circadian
plasma
of
=
D
D
DOC
75
O
*
4
i
o —
\h
1
iI
2
3
l
8°° 18°° 5. DAY
5
A
DAY OF TREATMENT
Fig.
il
2.
UAER and PAC in rats drinking saline or treated with DOC. NaCl: Rats received 0.9 °/o saline as drinking water for 5 days. DOC: Rats received a single dose of deoxycorticosterone oenanthate (2 mg/kg sc) on the 1st day of the experiment. PAC was measured in rats sacrificed at 08.00 or at 18.00 on the 5th day of the experiment. Values are means + sem (N 6). =
136
300-
200-
W §í 100
-aefíC 1
28
35
39 DAYS
-^-Hn
F
ï5 3 0.01. b) P < 0.01. c) P < 0.001
as
compared
to the
corresponding
Fig.
control.
4.
UAER, PAC and plasma corticosterone concentration in
rats treated with ACTH. dissolved in 0.5 ml of isotonic laevulose solution and injected into a tail vein at 09.00. Controls received 0.5 ml of vehicle only. Urine was collected for the following 8 h and blood was collected 1 h after treatment. Plasma concentrations of aldosterone and corticosterone were measured in the same animals. Values represent means ± sem (N 6).
/J1-24-ACTH
was
=
138
Fig.
5.
PAC and UAER in rats treated with a single dose of furosemide (25 mg/kg sc) (Fur) at the start of the experiment (09.00). For measurement of PAC groups of rats were sacrificed at the times indicated. For comparison the 24 h-course of PAC in control rats (Co) is given (see Fig. 1). For determination of UAER urine was collected for 24 h from rats treated with furosemide or ¡sotonic laevulose at 09.00. Values represent means + sem (N 6). Solid black abscissa indicates time of darkness. =
In rats kept on sodium-poor diet for 5 weeks (Fig. 3) urinary sodium ex¬ cretion declined below 1/200 of the controls within 3 days returning to control levels within 3 days after re-feeding the standard diet. UAER gradually in¬ creased to levels 60 times about controls during 3 weeks and remained that high for the following 2 weeks. After re-feeding of standard diet UAER rapidly decreased to nearly normal values within 3 days. PAC increased more than 100-fold after 2 weeks of sodium restriction (Table 1). Within 4 days of re-feeding the standard diet PAC reached control values, while UAER remained slightly, but significantly (P
