Effect of Acute Potassium Loading on Plasma Renin and on Urinary Aldosterone in Rats P. CORVOL, M. E. ORLIN, P. DEGOULET, P H . FRESSINAUD, AND J. MENARD 1NSERM, U-36, 17 rue de Fer a Moulin, 75005 Paris, France Potassium induced a natriuresis which was correlated directly with the dose of potassium administered (r = 0.89, P < 0.001). Potassium loading also increased plasma renin concentration which was correlated with the sodium excretion rate (r = 0.64, P < 0.01). Prevention of a negative sodium balance during the 769 fx,eq potassium load was obtained by administration of 513 /xeq sodium. In this experiment, plasma renin concentration increased little, whereas the aldosterone excretion rate was as high as during the 769 /xeq potassium load without sodium addition. (Endocrinology 100: 1008, 1977)
ABSTRACT. The disposition of aldosterone radiometabolites in rats has been studied following iv [3H]aldosterone administration. Of injected [3H]aldosterone, 0.31% is recovered in 24 h urine as free aldosterone and 0.08% as acid-labile conjugate. A simple, sensitive and reliable radioimmunoassay of free aldosterone has been developed and the effect of acute oral potassium loading (171, 513 or 769 /i.eq of KC1/100 g body weight) on 4 h aldosterone excretion, plasma renin concentration and sodium and potassium balance has been investigated. There was a positive correlation between log urinary aldosterone and potassium load (r = 0.92, P < 0.001).
V
ERY few physiological studies have been performed in vivo on aldosterone regulation in rats (1,2), due to the difficulty of measuring aldosterone in small biological samples and to an increase of aldosterone during stress and anesthesia (3,4). Therefore, the measurement of aldosterone in rat urine seemed to be a suitable alternative to the measurement of aldosterone in plasma. The purpose of this study was: 1) to determine the physiological disposition of aldosterone metabolites in order to evaluate what percentage of [3H]aldosterone was excreted as free and as acid-labile conjugate, 2) to develop a radioimmunoassay for free urinary aldosterone, and 3) to evaluate the effect of potassium, a known stimulus of aldosterone secretion, on free urinary aldosterone excretion. The results confirm that this method is a suitable tool for aldosterone physiological studies in rats. Materials and Methods In all experiments, male Wistar rats, weighing approximately 200 g, were trained for gentle handling. [l,2-3H]Aldosterone (50 Ci/mmol) Received January 22, 1976. This work has been partially supported by grants from INSERM.
was obtained from the Centre de l'Energie Atomique (Saclay, France) and its purity checked before use as already described (5). [4-14C]Aldosterone (50 mCi/mmol) was purchased from New England Nuclear Corp. Non-radioactive aldosterone was obtained from Sigma. All solvents were spectrograde quality and were purchased from Merck. 24 h disposition of urinary radiometabolites
[3H ]aldosterone
Rats were injected in the jugular vein with 10 fid of [3H]aldosterone previously dissolved in 0.9% NaCl containing 5% ethanol. The rats were then immediately placed in metabolic cages and urine and feces were collected separately for 24 h. During this time, the rats received 10 ml of 10% glucose in tap water. Specimens of urine and feces were analyzed for total radioactivity content and for identification of urinary [3H]aldosterone as free aldosterone and as acid-labile metabolite. Urine was adjusted to pH 7.0 and 5,000 dpm of [14C]aldosterone and 200 jug carrier aldosterone were added in order to correct for internal losses. Urine was extracted with two volumes of dichloromethane. The CH2C12 extract was washed with one volume of NaOH (0.1N) and one volume of distilled water and chromatographed in a Bush 5 system (benzene, methanol, water: 100, 50, 50). At the end of the chromatography the aldosterone zone was eluted with CH2C12, and one aliquot
1008
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POTASSIUM EFFECT ON RENIN-ALDOSTERONE was counted and another aliquot was acetylated. The aldosterone acetate so prepared was chromatographed in a Bush system (cyclohexane, benzene, methanol, water: 100, 40, 100, 20) and then by thin layer chromatography on silica gel (benzene, ethanol: 93, 7). After each chromatography, the 3 H and 14C were counted. Radiochemical purity was demonstrated by a variation of less than 5% in the 3H/14C ratio during the last two chromatographies. The aqueous phase obtained after dichloromethane extraction was hydrolyzed with HC1 (1.0 N) at pH 1.0 for 24 h at room temperature (6). After hydrolysis the acid-labile conjugate of aldosterone was extracted with CH2C12 and chromatographed exactly as for free aldosterone. The residual nonorganic phase will be referred to as "aqueous residue." The feces and total radioactive metabolites were dried in an oven at 37 C for 4 days, weighed, and ground in a mortar. Four aliquots were combusted in a tissue oxidizer (Oxymat— Intertechnique). Counting was performed in a Packard Tri Carb Liquid scintillation spectrometer. All counts were corrected for quenching and the spillover of 14C into the 3H channel (25%) was subtracted. Urinary aldosterone
radioimmunoassay
Urinary free aldosterone was extracted with 2 volumes of CH2C12. After washing with one volume of NaOH (O.IN) and one volume of water, the extract was directly submitted to radioimmunoassay. The high degree of specificity and sensitivity of the 3-carboxymethoxy-aldosterone
1009
diacetate antiserum used has been detailed (7) and the procedure of the radioimmunoassay is exactly the same as that already described (7). The specificity of the urinary radioimmunoassay was established by comparing the results obtained in 8 samples by the direct method to those obtained by paper chromatography, as previously described (7). Excretion of urinary free aldosterone under water loading was determined as follows: after 18 h of fasting, 11 rats were orally loaded at 0800 h with 5 ml of water/100 g body weight. The urine of each rat was then collected for the next 4 h, and urinary aldosterone was expressed as ng of free aldosterone excreted in 4 h. Also, urine of four adrenalectomized rats was individually collected for 24 h and assayed for aldosterone. Effect of acute potassium loading on the reninangiotensin aldosterone system Rats were fasted 18 h before the day of the experiment. On the day of the experiment, each rat was weighed and was then given at 0800 h by gastric administration distilled water (5 ml/100 g BW) containing 171, 513 or 769 fxeq of potassium (KC1). In another series of experiment, 513 /ieq of sodium (NaCl) were added to 769 /u,eq of potassium (KC1). The rats were then put immediately into individual metabolic cages for urine collection over the next 4 h, at the end of which rats were anesthetized with ether and jugular venous blood was collected in heparin. Sodium and potassium concentrations were measured by flame photometry for determination of sodium and potassium excretion rates (UNaV
TABLE 1. Fractionation of [ 3 H]aldosterone radiometabolites in urine
Rat no. 1 2 3 4
5 Mean ± SD
Total directly extracted by CH2C12* 13.2 14.5 13.6 11.7 10.3 12.7 ± 1.7
Not extracted initially by CH2CI2 but extracted after HC1*
Aqueous residue*'**
Free aldosterone t
6.3 5.3 6.7 6.3
68.2 63.4 67.9 76.4
6.2 ± 0.6
68.9 ± 5.4
0.26 0.31 0.30 0.40 0.27 0.31 ± 0.05
Acid-labile aldosterone conjugate f 0.06 0.08 0.08 0.10 0.08 ± 0.02
* Per cent of the total urinary radioactivity. Internal losses were corrected by addition of [ I4 C]aldosterone. ** Aqueous residue: Radioactivity not extracted initially by CH2C12) and after hydrolysis at pH 1.0 still not extracted by CH2C12. t Per cent of the total radioactivity injected.
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Endo • 1977 Vol 100 • No 4
CORVOL ET AL.
1010
and UKV). Sodium and potassium balances were calculated, by subtracting UNaV and UKV from the known amounts of sodium and potassium given orally. Plasma renin concentration (PRC) was measured according to the technique of Menard and Catt (8). Protein concentration was measured by the Biuret method (9). Results were expressed as mean ± 1 SD. The least squares method was used for the calculation of the regression coefficient and the slope of the regression line. Variance analysis was performed to verify the linearity of each regression line (10). Results 1. Urinary disposition of [3H]aldosterone radiometabolites In five rats, 29.9 ± 4.7% of the total radioactivity was excreted in urine, whereas 40.0 ± 11.9% was recovered in feces. Fractionation of the [3H]aldosterone radiometabolites in urine showed that about 13% of the total urinary radioactivity was directly extracted by CH2C12 and that an additional 6% of this radioactivity could be extracted after hydrolysis by HC1 (Table 1). Table 1 shows that 0.3% of the total radioactivity injected was recovered as free aldosterone and less than 0.1% as an acid-labile conjugate of aldosterone. 2. Urinary free aldosterone radioimmunoassay Since rat urine contained a significant fraction of free aldosterone, it was decided
to perform the urinary aldosterone radioimmunoassay on free, non-metabolized aldosterone. The specificity of the radioimmunoassay has been established on 8 different samples by measuring aldosterone on non-purified extracts and after paper chromatography. There was an excellent correlation between these two methods (r = 0.96, P < 0.001). The linear regression analysis gave the following equation: ng of aldosterone by chromatography = 1.17 + 0.96 x ng of aldosterone without chromatography. There was no systematic error in the method and the intercept of the regression line with the y axis was close to zero. Solvent blanks and water blanks were indistinguishable from zero. Urine from 4 adrenalectomized rats were also indistinguishable from zero. The inter and intra-assay reproducibility of the method was, respectively, 12 and 8%. Aldosterone excretion in 11 rats given a water load was 1.79 ± 0.46 ng/4 h. 3. Effect of acute potassium loading on renin-angiotensin aldosterone system a). Relation between potassium load and potassium balance and urinary aldosterone excretion. Data of potassium balance, plasma potassium, UNaV, PRC and aldosterone excretion are detailed in Table 2. There is a linear relationship between the logarithm of urinary aldosterone and the dose of potassium given (r = 0.919,
TABLE 2. Data for rats subjected to potassium loading and studied for a 4 hour period
Load* 171 /ueq Potassium 513 /aeq Potassium 769 pieq Potassium 769 /xeq Potassium + 513/xeq . Sodium
n
Potassium balance** jieq/100 g/4 h
Plasma potassium meq/1
Plasma protein g/1
7
50.6 ± 24.7
4.07 ± 0.27
7
123.4 ± 77.9
6
7
u Na vg/4 h /ieq/100
PRCf ng Angiotensin I /ml/2 h
Urinary free aldosterone ng/4h
59.71 ± 2.29
52.6 ± 30.8
88 ± 3 9
2.70 ± 0.72
4.23 ± 0.25
66.43 ± 0.98
234.3 ± 92.4
156 ± 55
6.67 ± 1.59
162.0 ± 62.1
5.01 ± 0.29
66.67 ± 3.44
343.8 ± 51.6
168 ± 40
10.25 ± 2.53
176.9 ± 41.5
4.30 ± 0.25
63.71 ± 1.50 538.1 ±45.2
64.4 ± 40.3
11.27 ± 3.79
* Potassium (KC1) and sodium (NaCl) loading are expressed in fieq/100 g body weight. ** Potassium balance is the difference between input and output, t PRC: Plasma renin concentration.
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POTASSIUM EFFECT ON RENIN-ALDOSTERONE P < 0.001) (Fig. 1). The extrapolation to the ordinate, corresponding to a zero potassium dose, gave a value of 1.84 ng of aldosterone excreted in 4 h. This value compares very well to the value of 1.79 found in waterloaded rats. There is also a significant correlation between the positive change in potassium balance and log urinary aldosterone (r = 0.596, P < 0.01) (Fig. 2). Finally a correlation was also found between log urinary aldosterone and plasma potassium (r = 0.560, P < 0.02). b). Relation between potassium load and sodium excretion. One of the most striking effects of the potassium load was to induce a natriuresis dependent on the amount of potassium loading. There is a linear relation between UNaV and K+ load (r = 0.894, P < 0.001).
1011 Aldo= 3.112 > 1.00502*
balance
r, 0.596 p 0.05). PRC was significantly lower than in rats given 769 and even 513 fieq potassium (P < 0.01) but the aldosterone excretion rate was as high as in rats given 769 /u,eq potassium only. Moreover, it is interesting to note that there was a significant difference between the plasma potassium levels on the highest potassium intake with and without sodium 200 400 600 800 loading (P < 0.001). The increased aldoPOTASSIUM LOAD sterone secretion acts in conjunction with FIG. 1. Relationship between the excretion of urinary the increased availability of sodium at the free aldosterone and the oral potassium load. Potas- cationic exchange site of the distal tubule sium load is expressed in /xeq/lOO g body weight. Points are the mean of 7 animals and the brackets to potentiate urinary potassium excretion. As a result, plasma potassium decreases are the SD. KLoad
=
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1012
CORVOL ET AL.
during the association of sodium and potassium loading. Moreover, the volume expansion induced by sodium loading could contribute to a decreased plasma potassium level. Discussion This study shows that after iv administration of [3H]aldosterone about 30% of the total radioactivity is excreted in 24 h urines and 40% of the radioactivity is recovered in feces, which is in good agreement with the study of McCaa and Sulya (11). Approximately 0.3% of the administered aldosterone is excreted as free aldosterone and 0.08% as acid labile conjugate. It is possible, although not demonstrated in this study, that most of this latter compound consists of aldosterone-18-oxo-glucuronide, the acid labile conjugate of aldosterone, as it has been first described by Bougas et al. in humans (6). It is interesting to note that the fraction of aldosterone excreted as free aldosterone versus acid-labile metabolite is higher in rats than in humans. Since in humans the kidney is believed to be one of the main organs of production of 18-oxoglucuronide (12) it is possible that the rat kidney does not conjugate aldosterone at the same rate as the human kidney. Several difficulties are encountered with plasma aldosterone measurements in rats. Besides the alteration of the aldosterone response to angiotensin II induced by stress and anesthesia (3), changes in plasma volume induced by bleeding make serial determinations difficult in the same animals. Urinary steroid excretion has the advantage of integrating fluctuations of the plasma steroid and of allowing serial determinations in the same animal. The use of a highly specific antibody allows a direct determination of aldosterone after solvent extraction as has already been reported in human urine (13) and plasma (7,14). It is difficult to compare the free urinary aldosterone excretion found in this study to other aldosterone determinations performed
Endo • 1977 Vol 100 • No 4
in rats. By using double isotopic dilution method, Schwartz and Bloch (15) have reported that rats submitted to a dietary intake of 2 meq Na+ excreted 20 to 56 ng of aldosterone extractable at pH 1.0 in 24 h urine. This is in agreement with our studies where rats given a water load excreted about 2 ng of free aldosterone in 4 h, corresponding to 12 ng/24 h. Such an excretion rate would correspond to a secretion of 4 /ug/24 h. This value is in reasonably good agreement with the data extrapolated from the study of Bojesen (1). This author found in rats an aldosterone metabolic clearance rate of 18 ml/min and a plasma aldosterone concentration varying from 5 to 150 ng/100 ml, depending on whether the sodium diet was high or low. The aldosterone secretion rate calculated from these values thus ranged from 1.5 to 45 /xg/24 h. Potassium loading is a well known stimulus of aldosterone secretion in several species (16-18). However only two studies have shown an in vivo effect of potassium on aldosterone in rats (1,19). The present study demonstrates also an aldosterone stimulating effect of potassium, but several mechanisms could account for the increase of aldosterone excretion. A direct influence of potassium loading, potassium balance, or plasma potassium on aldosterone excretion is suggested by the high correlations found between these values, and is in excellent agreement with the direct action of potassium on the production of aldosterone during in vitro incubations of rat adrenal slices (19,20). However, besides the direct effect of potassium, the wellknown natriuretic effect (21-23) of acute potassium loading, also observed in this study, could contribute to the increase in aldosterone excretion, via stimulation of the renin-angiotensin system. The direct role of potassium on aldosterone excretion has been evaluated by giving to rats both a potassium load and a sodium load in order to prevent a negative sodium balance. The increase in PRC was effectively prevented but the aldosterone
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POTASSIUM EFFECT ON RENIN-ALDOSTERONE excretion rate was still similar to the values obtained with the same potassium load without the sodium addition. This suggests that potassium in rats is able to stimulate aldosterone independently of sodium losses and of angiotensin stimulation. Acknowledgments The authors gratefully acknowledge the technical assistance of Mses I. Laboulandine and M. F. Gonzales and A. Michaud.
References 1. Bojesen, E.,EurJ Steroids 1: 145, 1966. 2. Campbell, W. B., W. A. Pettinger, K. Keeton, and S. N. Brooks, / Pharmacol Exp Ther 193: 166, 1975. 3. Campbell, W. B., S. N. Brooks, and W. A. Pettinger, Science 184: 994, 1974. 4. Pettinger, W., A. Tanaka, K. Keeton, W. B. Campbell, and S. N. Brooks, Proc Soc Exp Biol Med 148: 625, 1975. 5. Corvol, P., X. Bertagna, and J. Bedrossian, Ada Endocrinol (Kbh) 75: 756, 1974. 6. Bougas, J., C. Flood, B. Little, J. F. Tait, S. A. S. Tait, and R. Underwood, In Baulieu, E. E., and P. Robel (eds.), Aldosterone, Blackwell, Oxford, 1964, p. 25. 7. Pham Huu Trung, M. T., and P. Corvol, Steroids 24: 587, 1974.
1013
8. Menard, J., and K. J. Catt, Endocrinology 90: 422, 1972. 9. Gornall, A. C , C. J. Bardawill, and M. M. David, J Biol Chem 177: 751, 1949. 10. Armitage, P., Statistical Methods in Medical Research, Wiley, New York, 1971, p. 270. 11. McCaa, C. S., and L. Sulya, Endocrinology 79: 815, 1966. 12. Luetscher, J. A., E. W. Hancock, C. A. Camargo, A. J. Dowdy, and C. W. Nokes,/ Clin Endocrinol Metab 38: 628, 1965. 13. Langan, J., R. Jackson, E. V. Adlin, and B. J. Channick J Clin Endocrinol Metab 38: 189, 1974. 14. McKenzie, J. K., and J. A. Clements,/ Clin Endocrinol Metab 38: 622, 1974. 15. Schwartz, J., and R. Bloch, Ann Endocrinol (Paris) 25: 113, 1964. 16. Williams, G. H., and R. G. Dluhy, Am J Med 53: 595, 1972. 17. Funder, J. W., J. Blair-West, J. P. Coghlan, D. A. Denton, B. A. Scoggins, and R. D. Wright, Endocrinology 85: 381, 1969. 18. Davis, J. O., J. Urquhart, and J. T. Higgins, Jr., J Clin Invest 42: 597, 1963. 19. Boyd, J. E., W. P. Palmore, and P. J. Mulrow, Endocrinology 88: 556, 1971. 20. Boyd, J. E., and P. J. Mulrow, Endocrinology 90: 299, 1972. 21. Brandis, M., J. Keyes, and E. E. Windhager, Am] Physiol 222: 421, 1972. 22. Kahn, M., and N. K. Bohrer, Am J Physiol 212: 1365, 1967. 23. Winkler, A. W., and P. K. Smith, Am J Physiol 138: 94, 1942.
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ABSTRACT. The disposition of aldosterone radiometabolites in rats has been studied following iv [3H]aldosterone administration. Of injected [3H]aldosterone, 0.31% is recovered in 24 h urine as free aldosterone and 0.08% as acid-labile conjugate. A simple, sensitive and reliable radioimmunoassay of free aldosterone has been developed and the effect of acute oral potassium loading (171, 513 or 769 /i.eq of KC1/100 g body weight) on 4 h aldosterone excretion, plasma renin concentration and sodium and potassium balance has been investigated. There was a positive correlation between log urinary aldosterone and potassium load (r = 0.92, P < 0.001).
V
ERY few physiological studies have been performed in vivo on aldosterone regulation in rats (1,2), due to the difficulty of measuring aldosterone in small biological samples and to an increase of aldosterone during stress and anesthesia (3,4). Therefore, the measurement of aldosterone in rat urine seemed to be a suitable alternative to the measurement of aldosterone in plasma. The purpose of this study was: 1) to determine the physiological disposition of aldosterone metabolites in order to evaluate what percentage of [3H]aldosterone was excreted as free and as acid-labile conjugate, 2) to develop a radioimmunoassay for free urinary aldosterone, and 3) to evaluate the effect of potassium, a known stimulus of aldosterone secretion, on free urinary aldosterone excretion. The results confirm that this method is a suitable tool for aldosterone physiological studies in rats. Materials and Methods In all experiments, male Wistar rats, weighing approximately 200 g, were trained for gentle handling. [l,2-3H]Aldosterone (50 Ci/mmol) Received January 22, 1976. This work has been partially supported by grants from INSERM.
was obtained from the Centre de l'Energie Atomique (Saclay, France) and its purity checked before use as already described (5). [4-14C]Aldosterone (50 mCi/mmol) was purchased from New England Nuclear Corp. Non-radioactive aldosterone was obtained from Sigma. All solvents were spectrograde quality and were purchased from Merck. 24 h disposition of urinary radiometabolites
[3H ]aldosterone
Rats were injected in the jugular vein with 10 fid of [3H]aldosterone previously dissolved in 0.9% NaCl containing 5% ethanol. The rats were then immediately placed in metabolic cages and urine and feces were collected separately for 24 h. During this time, the rats received 10 ml of 10% glucose in tap water. Specimens of urine and feces were analyzed for total radioactivity content and for identification of urinary [3H]aldosterone as free aldosterone and as acid-labile metabolite. Urine was adjusted to pH 7.0 and 5,000 dpm of [14C]aldosterone and 200 jug carrier aldosterone were added in order to correct for internal losses. Urine was extracted with two volumes of dichloromethane. The CH2C12 extract was washed with one volume of NaOH (0.1N) and one volume of distilled water and chromatographed in a Bush 5 system (benzene, methanol, water: 100, 50, 50). At the end of the chromatography the aldosterone zone was eluted with CH2C12, and one aliquot
1008
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POTASSIUM EFFECT ON RENIN-ALDOSTERONE was counted and another aliquot was acetylated. The aldosterone acetate so prepared was chromatographed in a Bush system (cyclohexane, benzene, methanol, water: 100, 40, 100, 20) and then by thin layer chromatography on silica gel (benzene, ethanol: 93, 7). After each chromatography, the 3 H and 14C were counted. Radiochemical purity was demonstrated by a variation of less than 5% in the 3H/14C ratio during the last two chromatographies. The aqueous phase obtained after dichloromethane extraction was hydrolyzed with HC1 (1.0 N) at pH 1.0 for 24 h at room temperature (6). After hydrolysis the acid-labile conjugate of aldosterone was extracted with CH2C12 and chromatographed exactly as for free aldosterone. The residual nonorganic phase will be referred to as "aqueous residue." The feces and total radioactive metabolites were dried in an oven at 37 C for 4 days, weighed, and ground in a mortar. Four aliquots were combusted in a tissue oxidizer (Oxymat— Intertechnique). Counting was performed in a Packard Tri Carb Liquid scintillation spectrometer. All counts were corrected for quenching and the spillover of 14C into the 3H channel (25%) was subtracted. Urinary aldosterone
radioimmunoassay
Urinary free aldosterone was extracted with 2 volumes of CH2C12. After washing with one volume of NaOH (O.IN) and one volume of water, the extract was directly submitted to radioimmunoassay. The high degree of specificity and sensitivity of the 3-carboxymethoxy-aldosterone
1009
diacetate antiserum used has been detailed (7) and the procedure of the radioimmunoassay is exactly the same as that already described (7). The specificity of the urinary radioimmunoassay was established by comparing the results obtained in 8 samples by the direct method to those obtained by paper chromatography, as previously described (7). Excretion of urinary free aldosterone under water loading was determined as follows: after 18 h of fasting, 11 rats were orally loaded at 0800 h with 5 ml of water/100 g body weight. The urine of each rat was then collected for the next 4 h, and urinary aldosterone was expressed as ng of free aldosterone excreted in 4 h. Also, urine of four adrenalectomized rats was individually collected for 24 h and assayed for aldosterone. Effect of acute potassium loading on the reninangiotensin aldosterone system Rats were fasted 18 h before the day of the experiment. On the day of the experiment, each rat was weighed and was then given at 0800 h by gastric administration distilled water (5 ml/100 g BW) containing 171, 513 or 769 fxeq of potassium (KC1). In another series of experiment, 513 /ieq of sodium (NaCl) were added to 769 /u,eq of potassium (KC1). The rats were then put immediately into individual metabolic cages for urine collection over the next 4 h, at the end of which rats were anesthetized with ether and jugular venous blood was collected in heparin. Sodium and potassium concentrations were measured by flame photometry for determination of sodium and potassium excretion rates (UNaV
TABLE 1. Fractionation of [ 3 H]aldosterone radiometabolites in urine
Rat no. 1 2 3 4
5 Mean ± SD
Total directly extracted by CH2C12* 13.2 14.5 13.6 11.7 10.3 12.7 ± 1.7
Not extracted initially by CH2CI2 but extracted after HC1*
Aqueous residue*'**
Free aldosterone t
6.3 5.3 6.7 6.3
68.2 63.4 67.9 76.4
6.2 ± 0.6
68.9 ± 5.4
0.26 0.31 0.30 0.40 0.27 0.31 ± 0.05
Acid-labile aldosterone conjugate f 0.06 0.08 0.08 0.10 0.08 ± 0.02
* Per cent of the total urinary radioactivity. Internal losses were corrected by addition of [ I4 C]aldosterone. ** Aqueous residue: Radioactivity not extracted initially by CH2C12) and after hydrolysis at pH 1.0 still not extracted by CH2C12. t Per cent of the total radioactivity injected.
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Endo • 1977 Vol 100 • No 4
CORVOL ET AL.
1010
and UKV). Sodium and potassium balances were calculated, by subtracting UNaV and UKV from the known amounts of sodium and potassium given orally. Plasma renin concentration (PRC) was measured according to the technique of Menard and Catt (8). Protein concentration was measured by the Biuret method (9). Results were expressed as mean ± 1 SD. The least squares method was used for the calculation of the regression coefficient and the slope of the regression line. Variance analysis was performed to verify the linearity of each regression line (10). Results 1. Urinary disposition of [3H]aldosterone radiometabolites In five rats, 29.9 ± 4.7% of the total radioactivity was excreted in urine, whereas 40.0 ± 11.9% was recovered in feces. Fractionation of the [3H]aldosterone radiometabolites in urine showed that about 13% of the total urinary radioactivity was directly extracted by CH2C12 and that an additional 6% of this radioactivity could be extracted after hydrolysis by HC1 (Table 1). Table 1 shows that 0.3% of the total radioactivity injected was recovered as free aldosterone and less than 0.1% as an acid-labile conjugate of aldosterone. 2. Urinary free aldosterone radioimmunoassay Since rat urine contained a significant fraction of free aldosterone, it was decided
to perform the urinary aldosterone radioimmunoassay on free, non-metabolized aldosterone. The specificity of the radioimmunoassay has been established on 8 different samples by measuring aldosterone on non-purified extracts and after paper chromatography. There was an excellent correlation between these two methods (r = 0.96, P < 0.001). The linear regression analysis gave the following equation: ng of aldosterone by chromatography = 1.17 + 0.96 x ng of aldosterone without chromatography. There was no systematic error in the method and the intercept of the regression line with the y axis was close to zero. Solvent blanks and water blanks were indistinguishable from zero. Urine from 4 adrenalectomized rats were also indistinguishable from zero. The inter and intra-assay reproducibility of the method was, respectively, 12 and 8%. Aldosterone excretion in 11 rats given a water load was 1.79 ± 0.46 ng/4 h. 3. Effect of acute potassium loading on renin-angiotensin aldosterone system a). Relation between potassium load and potassium balance and urinary aldosterone excretion. Data of potassium balance, plasma potassium, UNaV, PRC and aldosterone excretion are detailed in Table 2. There is a linear relationship between the logarithm of urinary aldosterone and the dose of potassium given (r = 0.919,
TABLE 2. Data for rats subjected to potassium loading and studied for a 4 hour period
Load* 171 /ueq Potassium 513 /aeq Potassium 769 pieq Potassium 769 /xeq Potassium + 513/xeq . Sodium
n
Potassium balance** jieq/100 g/4 h
Plasma potassium meq/1
Plasma protein g/1
7
50.6 ± 24.7
4.07 ± 0.27
7
123.4 ± 77.9
6
7
u Na vg/4 h /ieq/100
PRCf ng Angiotensin I /ml/2 h
Urinary free aldosterone ng/4h
59.71 ± 2.29
52.6 ± 30.8
88 ± 3 9
2.70 ± 0.72
4.23 ± 0.25
66.43 ± 0.98
234.3 ± 92.4
156 ± 55
6.67 ± 1.59
162.0 ± 62.1
5.01 ± 0.29
66.67 ± 3.44
343.8 ± 51.6
168 ± 40
10.25 ± 2.53
176.9 ± 41.5
4.30 ± 0.25
63.71 ± 1.50 538.1 ±45.2
64.4 ± 40.3
11.27 ± 3.79
* Potassium (KC1) and sodium (NaCl) loading are expressed in fieq/100 g body weight. ** Potassium balance is the difference between input and output, t PRC: Plasma renin concentration.
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POTASSIUM EFFECT ON RENIN-ALDOSTERONE P < 0.001) (Fig. 1). The extrapolation to the ordinate, corresponding to a zero potassium dose, gave a value of 1.84 ng of aldosterone excreted in 4 h. This value compares very well to the value of 1.79 found in waterloaded rats. There is also a significant correlation between the positive change in potassium balance and log urinary aldosterone (r = 0.596, P < 0.01) (Fig. 2). Finally a correlation was also found between log urinary aldosterone and plasma potassium (r = 0.560, P < 0.02). b). Relation between potassium load and sodium excretion. One of the most striking effects of the potassium load was to induce a natriuresis dependent on the amount of potassium loading. There is a linear relation between UNaV and K+ load (r = 0.894, P < 0.001).
1011 Aldo= 3.112 > 1.00502*
balance
r, 0.596 p 0.05). PRC was significantly lower than in rats given 769 and even 513 fieq potassium (P < 0.01) but the aldosterone excretion rate was as high as in rats given 769 /u,eq potassium only. Moreover, it is interesting to note that there was a significant difference between the plasma potassium levels on the highest potassium intake with and without sodium 200 400 600 800 loading (P < 0.001). The increased aldoPOTASSIUM LOAD sterone secretion acts in conjunction with FIG. 1. Relationship between the excretion of urinary the increased availability of sodium at the free aldosterone and the oral potassium load. Potas- cationic exchange site of the distal tubule sium load is expressed in /xeq/lOO g body weight. Points are the mean of 7 animals and the brackets to potentiate urinary potassium excretion. As a result, plasma potassium decreases are the SD. KLoad
=
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1012
CORVOL ET AL.
during the association of sodium and potassium loading. Moreover, the volume expansion induced by sodium loading could contribute to a decreased plasma potassium level. Discussion This study shows that after iv administration of [3H]aldosterone about 30% of the total radioactivity is excreted in 24 h urines and 40% of the radioactivity is recovered in feces, which is in good agreement with the study of McCaa and Sulya (11). Approximately 0.3% of the administered aldosterone is excreted as free aldosterone and 0.08% as acid labile conjugate. It is possible, although not demonstrated in this study, that most of this latter compound consists of aldosterone-18-oxo-glucuronide, the acid labile conjugate of aldosterone, as it has been first described by Bougas et al. in humans (6). It is interesting to note that the fraction of aldosterone excreted as free aldosterone versus acid-labile metabolite is higher in rats than in humans. Since in humans the kidney is believed to be one of the main organs of production of 18-oxoglucuronide (12) it is possible that the rat kidney does not conjugate aldosterone at the same rate as the human kidney. Several difficulties are encountered with plasma aldosterone measurements in rats. Besides the alteration of the aldosterone response to angiotensin II induced by stress and anesthesia (3), changes in plasma volume induced by bleeding make serial determinations difficult in the same animals. Urinary steroid excretion has the advantage of integrating fluctuations of the plasma steroid and of allowing serial determinations in the same animal. The use of a highly specific antibody allows a direct determination of aldosterone after solvent extraction as has already been reported in human urine (13) and plasma (7,14). It is difficult to compare the free urinary aldosterone excretion found in this study to other aldosterone determinations performed
Endo • 1977 Vol 100 • No 4
in rats. By using double isotopic dilution method, Schwartz and Bloch (15) have reported that rats submitted to a dietary intake of 2 meq Na+ excreted 20 to 56 ng of aldosterone extractable at pH 1.0 in 24 h urine. This is in agreement with our studies where rats given a water load excreted about 2 ng of free aldosterone in 4 h, corresponding to 12 ng/24 h. Such an excretion rate would correspond to a secretion of 4 /ug/24 h. This value is in reasonably good agreement with the data extrapolated from the study of Bojesen (1). This author found in rats an aldosterone metabolic clearance rate of 18 ml/min and a plasma aldosterone concentration varying from 5 to 150 ng/100 ml, depending on whether the sodium diet was high or low. The aldosterone secretion rate calculated from these values thus ranged from 1.5 to 45 /xg/24 h. Potassium loading is a well known stimulus of aldosterone secretion in several species (16-18). However only two studies have shown an in vivo effect of potassium on aldosterone in rats (1,19). The present study demonstrates also an aldosterone stimulating effect of potassium, but several mechanisms could account for the increase of aldosterone excretion. A direct influence of potassium loading, potassium balance, or plasma potassium on aldosterone excretion is suggested by the high correlations found between these values, and is in excellent agreement with the direct action of potassium on the production of aldosterone during in vitro incubations of rat adrenal slices (19,20). However, besides the direct effect of potassium, the wellknown natriuretic effect (21-23) of acute potassium loading, also observed in this study, could contribute to the increase in aldosterone excretion, via stimulation of the renin-angiotensin system. The direct role of potassium on aldosterone excretion has been evaluated by giving to rats both a potassium load and a sodium load in order to prevent a negative sodium balance. The increase in PRC was effectively prevented but the aldosterone
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POTASSIUM EFFECT ON RENIN-ALDOSTERONE excretion rate was still similar to the values obtained with the same potassium load without the sodium addition. This suggests that potassium in rats is able to stimulate aldosterone independently of sodium losses and of angiotensin stimulation. Acknowledgments The authors gratefully acknowledge the technical assistance of Mses I. Laboulandine and M. F. Gonzales and A. Michaud.
References 1. Bojesen, E.,EurJ Steroids 1: 145, 1966. 2. Campbell, W. B., W. A. Pettinger, K. Keeton, and S. N. Brooks, / Pharmacol Exp Ther 193: 166, 1975. 3. Campbell, W. B., S. N. Brooks, and W. A. Pettinger, Science 184: 994, 1974. 4. Pettinger, W., A. Tanaka, K. Keeton, W. B. Campbell, and S. N. Brooks, Proc Soc Exp Biol Med 148: 625, 1975. 5. Corvol, P., X. Bertagna, and J. Bedrossian, Ada Endocrinol (Kbh) 75: 756, 1974. 6. Bougas, J., C. Flood, B. Little, J. F. Tait, S. A. S. Tait, and R. Underwood, In Baulieu, E. E., and P. Robel (eds.), Aldosterone, Blackwell, Oxford, 1964, p. 25. 7. Pham Huu Trung, M. T., and P. Corvol, Steroids 24: 587, 1974.
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8. Menard, J., and K. J. Catt, Endocrinology 90: 422, 1972. 9. Gornall, A. C , C. J. Bardawill, and M. M. David, J Biol Chem 177: 751, 1949. 10. Armitage, P., Statistical Methods in Medical Research, Wiley, New York, 1971, p. 270. 11. McCaa, C. S., and L. Sulya, Endocrinology 79: 815, 1966. 12. Luetscher, J. A., E. W. Hancock, C. A. Camargo, A. J. Dowdy, and C. W. Nokes,/ Clin Endocrinol Metab 38: 628, 1965. 13. Langan, J., R. Jackson, E. V. Adlin, and B. J. Channick J Clin Endocrinol Metab 38: 189, 1974. 14. McKenzie, J. K., and J. A. Clements,/ Clin Endocrinol Metab 38: 622, 1974. 15. Schwartz, J., and R. Bloch, Ann Endocrinol (Paris) 25: 113, 1964. 16. Williams, G. H., and R. G. Dluhy, Am J Med 53: 595, 1972. 17. Funder, J. W., J. Blair-West, J. P. Coghlan, D. A. Denton, B. A. Scoggins, and R. D. Wright, Endocrinology 85: 381, 1969. 18. Davis, J. O., J. Urquhart, and J. T. Higgins, Jr., J Clin Invest 42: 597, 1963. 19. Boyd, J. E., W. P. Palmore, and P. J. Mulrow, Endocrinology 88: 556, 1971. 20. Boyd, J. E., and P. J. Mulrow, Endocrinology 90: 299, 1972. 21. Brandis, M., J. Keyes, and E. E. Windhager, Am] Physiol 222: 421, 1972. 22. Kahn, M., and N. K. Bohrer, Am J Physiol 212: 1365, 1967. 23. Winkler, A. W., and P. K. Smith, Am J Physiol 138: 94, 1942.
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