Sodium balance and renin regulation in rats: Role of intrinsic renal mechanisms

Kidney International, Vol. 17 (1980), pp. 607-614

Sodium balance and renin regulation in rats: Role of intrinsic renal mechanisms HARUYUKI NAKANE, YoKo NAKANE, PIERRE CORVOL, and JOEL MENARD Inst itut National de Ia Sante et de Ia Recherche Médicale, Unite 36, Paris, France

Plasma renin levels and renal renin content vary according to the sodium balance of subjects or animals [1-4]. Usually, these two parameters change in parallel, and when sodium balance variation is prolonged, renal renin content appears to be one of the major determinants of renin release in response to various stimuli [5, 6]. The lack of a close link between renin content and plasma renin level, however, also has been demonstrated [5, 7, 8]. Sodium loading with deoxycorticosterone decreases plasma renin activity in animals as early as 24 to 48 hours after treatment begins [7, 9]. Sodium deprivation also induces a rapid increase in plasma renin levels [2,

Sodium balance and renin regulation in rats: Role of renal intrinsic mechanisms. Renin secretion was compared in vivo and in

vitro among five groups of rats, each group subjected to a different sodium balance for 10 to 14 days. The state of the reninangiotensin system in vivo was evaluated by measuring the renal renin (RR) and the plasma renin concentration (PRC) in both anesthetized and nonanesthetized animals. The in vitro renin secretion rate (RSR) was determined in isolated perfused kidneys. RR was reduced (—48%) by sodium loading and deoxycorticosterone (DOCA) and increased (+27%) by sodium deprivation and furosemide. Sodium loading and DOCA reduced both the PRC and the RSR to less than 20% of control values. By contrast, sodium deprivation and furosemide induced a more than fourfold rise in

the PRC but only a small increase in the RSR (+ 37%). These results indicate that changes in fractional renin release are induced by sodium balance variation, and these changes are preserved in vitro only in sodium-loaded states. The inability of sodium-deprived kidneys to maintain high renin release in vitro suggests that high plasma renin levels in these rats depend on mechanisms that are not preserved in vitro. There was no evidence supporting the participation of inactive renin secretion in the regulation of fractional renin release under varying sodium balance.

3]. In such acute sodium balance variation, the change in plasma renin levels is not accompanied by

a proportional change in renal renin content [7]. These findings suggest that the state of sodium balance determines the activity of certain mechanisms that regulate the proportion of stored renin mobilized from its pool. Although many intrarenal and extrarenal factors are known to influence renin release [2, 10], little is known about the essential factors regulating renin mobilization in different states of sodium balance. We undertook the present study to determine whether such regulation is mainly de-

Bilan du sodium et regulation de Ia rénine chez le rat: Role des

mécanismes rénaux intrinséques. La sécrétion de rénine a été comparCe in vivo et in vitro dans 5 groupes de rats soumis a différents apports de sodium pendant 10 a 14 jours. L'état du système refine angiotensine in vivo a été évaluC par Ia mesure de la refine renale (RR) et de Ia concentration plasmatique de rénine (PRC) chez des animaux anesthésiés et non anesthésies. Le debit de sécrétion de rénine in vitro (RSR) a Cté déterminé dans des reins isolés perfusés. RR a été rCduite (—48%) par Ia charge en sodium et Ia desoxycorticostérone (DOCA) et augmentée (+27%) par Ia restriction sodCe et le furosémide. La réduction de PRC et RSR due a Ia charge en sodium associée a la

pendent on extrarenal or intrarenal mechanisms. For this purpose, we compared circulating renin and renal renin in vivo and in vitro using rats maintained on different sodium regimens. To evaluate

DOCA a èté d'amplitude identique (moms de 20% des valeurs con-

the state of renin-angiotensin system in vivo, we

trôles). Par contre, la restriction sodée et Ic furosémide quadruplent PRC, mais n'augmentent que faiblement RSR (+37%). Ces résultats indiquent que des changements de Ia fraction de rénine

measured renal renin content and plasma renin con-

centration in anesthetized and nonanesthetized rats. The rat plasma samples were examined also

rénale sCcrétée sont entraInées par les variations du bilan de sodium. Ces changements sont preserves in vitro dans les situations de charge en sodium. Au contraire, après restriction sodée, le maintien in vitro d'une libération de rénine importante n'est pas possible et est donc entretenu in vivo par des mécanismes qui ne sont pas transférables in vitro. Par ailleurs, il n'y a pas d'argument en faveur de La participation d'une sécrétion de rénine inactive dans Ia regulation de Ia fraction de rénine rénale Iibérée lors de différentes conditions d'apports en sodium.

Received for publication January 9, 1979 and in revised form September 26, 1979 0085-2538/80/0017-0607 $0 1.60

© 1980 by the International Society of Nephrology

607

Nakane et a!

608

for the content of acid-activatable renin, to see if the secretion of such renin does exist in rats and if it is related to the state of sodium balance [11—13]. Then, to exclude the influence of extrarenal factors, we removed the kidneys from the animals and per-

fused them in vitro for determination of renin release under controlled conditions. Methods

Male Wistar rats, each weighing 330 to 400 g, were divided into five groups and were maintained on different regimens for 10 to 14 days. Group 1 rats (controls) were fed a regular diet (sodium content, 170 mEq/kg of food) and were given tap water to drink. Group 2 rats had a regular diet, 1% sodium chloride solution for drinking, and deoxycorticos-

terone trimethylacetate (DOCA), injected intramuscularly at a weekly dose of 8 mg/animal. Group 3 rats were fed the same diet as group 2 but were not given DOCA injections. Group 4 rats were fed a low sodium diet (sodium content, 4 mEq/kg of food) and

were given tap water to drink. Group 5 rats were treated like the rats in group 4, and were given sub-

cutaneous furosemide injections (2 mg/animal, twice a day) for 2 days preceding the experiment. The last furosemide injection was given 20 hours before the experiment. On the day of the experiment, some of the animals from groups 1, 3, and 5 were killed by decapitation, and their kidneys and plasma were taken for measurements of renal renin

(RR), plasma renin concentration (PRC), and plasma acid-activatable renin. Other animals were used for perfusion experiments. Kidney perfusion in vitro. The perfusion methods

were mostly those described by Bowman and Maack [14] with recent modifications [15]. While the rats were under pentobarbital anesthesia (40 to 50 mg/kg, i.p.), blood samples (0.6 to 1.0 ml) were collected for PRC measurement before other operative procedures began. Then, after the ureter was catheterized, the right kidney was cannulated, iso-

lated, and placed in an oxygenator chamber in which 80 ml of recirculating medium was aerated by

oxygen and carbon dioxide (95:5). A blood-free Krebs-Ringer bicarbonate buffer containing 5.5 m glucose and 5% bovine serum albumin (Cohn's Fraction V, Sigma) was used for the perfusate.

Hg (mean pressure). A 20-mm equilibration period was allowed before the 1-hour observation began,

to avoid the initial functional instability of the perfused kidney. The PP and perfusate flow were determined by previously described methods [15]. Urine and perfusate were sampled every 15 mm for clearance measurements. Changes in perfusate volume and in sodium and potassium concentrations due to sampling and urinary loss were corrected as previously described [15]. The renin concentration in the perfusate was measured in samples taken at 0, 30, and 60 mm of the observation period. After the perfusion ended, the kidney was weighed and stored frozen. Renin assay: (1) Plasma and perfusate samples. Renin concentration was measured by radioimmu-

noassay of angiotensin I (Al) generated during 2 hours of incubation with a homologous substrate according to previously described methods [16]. All

measurements were performed in duplicate or quadruplicate. The renin concentration was expressed by the quantity of Al formed by a 1-mI sample in 1 hour at 37° C in the presence of an excess of renin substrate. Acid-activatable renin was measured in plasma samples from nonanesthetized rats by the following methods: a portion of each plasma (0.5 to 1.0 ml) was dialyzed to a pH of 3.3 against a hydrogen chloride and L-glycine buffer (0.2 M) for 24 hours at 4° C, and then redialyzed to a pH of 7.4 against a phosphate buffer (0.2 M) for another 24 hours at 4° C. Another portion of each plasma was dialyzed to a pH of 7.4 against a phosphate buffer (0.2 M) for 48 hours at 4° C and was used as the dialyzed control. EDTA (10 mM) was added to all dialysates. Renm concentration in dialyzed samples was determined by previously described methods [17]. (2) Kidney samples. Kidneys were kept frozen at —20° C until processed. Each kidney was homogenized in a Potter apparatus at 0° C in 0.2 M phosphate buffer (pH, 6.5). After freezing and thawing

four times, the homogenates were centrifuged at x 3200g for 20 mm, and the supernatant was measured for renin concentration and protein content. Incubation conditions and the Al radioimmunoassay

were the same for the renal renin and plasma and perfusate renin assays. Renal renin content (RR)

To this, tritiated methoxyinulin (10 MCi/liter) (New

was expressed by micrograms of A! per hour1

England Nuclear) was also added to measure the GFR. The left kidney was taken out and stored frozen. The kidney in the oxygenator chamber was perfused by a peristaltic pump at a perfusion pres-

per milligram1 of protein. Other analyses. The rate of renin secretion in vitro was calculated according to the increase in perfusate renin concentration per time unit multiplied by the perfusate volume and was expressed by mi-

sure (PP) maintained within a range of 65 to 80 mm

Sodium balance and renin regulation

C

C

** *

C

0 NJ

** *

0

0

*

C,,

.1-

a,

***

_____________

0 C., =

PRC in nonanesthetized rats, og of Al -hr

a,

____________________________________I—

*

C

C

L

C 0

PRC in anesthetized rats, fly of Al-hr

-

609

Groupi

3

2

(N = 21)

(N=

4

(N 7) 14)

(N= 6(

5 (N = 15(

2

1

3

4

5

1

(N = 10) (N= 7) (N= 6) (N= 9(

(N = 15)

(N= 6)

2 (N = 5)

5

(N= 5)

Fig. 1. Renin secretion in vivo: Renal renin content and plasma renin concentration (PRC) in various states of sodium balance. Mean values (bars) and I SEM (small vertical lines) are shown. Darker bars indicate groups with greater sodium intake. Group numbers are

indicated under each bar. The numbers in parentheses are numbers of animals. Note that the scale of PRC for anesthetized and nonanesthetized rats is not identical for graphic reasons. Probability values are calculated vs. the value in group I (controls) and are indicated by asterisks: <0.05, **p <0.01, ***p <0.001.

crograms of Al per minutes-1. This value was

For statistical analysis, standard methods were

thought to represent the amount of renin secreted by the perfused kidney, because in preliminary experiments with purified hog renin [18], perfused rat kidneys did not degrade appreciably the renin added to the medium. To compare the amount of renin

used [20]. P values less than 0.05 were accepted as significant.

secreted in vitro with the renal renin content in each

animal, we calculated the fractional renin release

(FRR) from the expression renin secretion rate times 60 mm divided by the whole renin content in the left kidney. In this formula, the left kidney renin

content was considered to represent the preperfusion value of renal renin content. Tritiated methoxyinulin was measured by liquid scintillation counting with a spectrometer (Packard

Tricarb, model 3380). Sodium was measured by flame photometry (Electro-Synthèse). Urine volume was determined gravimetrically. Protein content was measured by the method of Lowry et al [19]. All results are expressed as the means SEM.

Results

Weight gain in control rats (group 1, N = 21) was 2.7 0.2 g . day. Sodium-loaded rats (groups 2

and 3, N =

and 7, respectively) gained less weight than did control rats (1.9 0.2 g day-'). Sodium-deprived rats (groups 4 and 5, N = 6 and 14

15, respectively) slightly lost weight (—0.9 0.4 g 1.4 g of weight during the last 24 hours due to the effect of furosemide.

day-'). GroupS rats lost 11.5

Renin secretion in vivo and in vitro. Figure 1 presents the results obtained in vivo. These data confirmed the well-known observations that plasma renin levels rise during sodium deprivation and diminish during sodium loading. Compared with other

groups, the difference from control values was

Nakane et a!

610

greater when the variation in sodium balance was

renin compared with the control group. Renin content in the right perfused kidneys (r-RR) measured after each experiment was well correlated with the renin content in the corresponding left, nonperfused kidney (l-RR) (r = 0.91, P < 0.001). When the renin content was compared between each kidney pair, rRR was significantly smaller than l-RR in groups 1, 4, and 5 by 10 to 20%. The difference in renin content between each kidney pair was correlated with the amount of renin found in the final perfusate (r = 0.53, P < 0.001). The sum of r-RR and the renin in the final perfusate did not usually exceed the corresponding l-RR (in 37 out of 46 kidney pairs), and this tendency was not different between sodiumloaded and sodium-deprived groups. The evidence for de novo synthesis during perfusion was not supplied by these data. Rates of renin secretion in vitro (RSR) and the fractional renin release (FRR) calculated for the 1-hour perfusion are shown by Fig. 2. The most striking observation in vitro was the suppression of renin release in sodium-loaded groups, especially in group 2 kidneys. In sodium-deprived groups, RSR was only slightly higher than that in

reinforced by drugs (groups 2 and 5). In nonanesthetized rats, the PRC values were fourfold to fivefold less than those measured under anesthesia in keeping with previous reports [9, 21]. The relative PRC difference between the control and experimental groups was similar in anesthetized and in nonanesthetized rats. These data indicate obvious differences in magnitude between changes in RR and PRC. Compared with RR variations, the PRC varied in a far greater range both in anesthetized and nonanesthetized animals. Acidification of the plasma from nonanesthetized rats resulted in no increase in renin concentration, indicating the absence of acid-activatable renin from

the rat plasma. The A! formed by acid-dialyzed plasma was slightly more (+6.7 3.3%, P < 0.025, paired t test) than that by controls dialyzed to a pH of 7.4, but when compared with predialysis values, renin concentrations in acid-dialyzed samples were not elevated. The difference in renin concentration between acid-dialyzed plasma and plasma dialyzed to a pH of 7.4 was therefore attributed to the lower renin recovery at the pH of 7.4 than at the pH of 3.3, as recently demonstrated [17]. Other data not shown in Fig. 1 are the weights and protein contents of the kidneys. The kidneys from sodium-loaded rats were heavier (1.31 0.03 and 1.14 0.05 g in groups 2

the control group. There were no differences in FRR between the control and sodium-deprived groups. The relationship between PRC and RSR in

animals in different states of sodium balance is graphically presented by Fig. 3. When the data for groups 1, 2, and 3 (control + sodium-loaded rats) are compared (Fig. 3a), PRC is significantly correlated with RSR, whereas PRC and RSR are not correlated for groups 1, 4, and 5 (control + sodiumdeprived rats, Fig. 3b). Table 2 shows the other functional data for isolated perfused kidneys. The values are in agreement with previous reports [14, 15, 17]. Although some intergroup difference was noted in certain variables measured, the range of their variation was large, and no variables were closely correlated to the state of renin secretion of perfused kidneys.

and 3, respectively) than those from control rats (1.12 0.03 g) or sodium-deprived rats (0.97 0.04 and 1.11 0.04 in groups 4 and 5, respectively). Re-

nal protein content ranged between 90 and 150 mg/kidney and was well correlated with the kidney net weight (r = 0.88, P <0.001). Table 1 and Fig. 2 summarize the renin secretion

by perfused kidneys. As shown in Table 1, renin concentration in the perfusate increased gradually with time. Throughout the experiment, kidneys from sodium-loaded rats secreted less and those from sodium-deprived rats secreted more amount of

Table 1. Renin secretion by isolated perfused kidneys'

Renin concen tration in the perfusate, ng A! .

Groups (1) (2) (3) (4) (5)

0 mm

Control (N = 14) NaC1 loading + DOCA (N =9) NaCI loading (N = 7) Sodium deprivation N = 6) Sodiumdeprivation +furosemide(TV =

a Values are the means b

P <0.001 vs. control.

d

P <0.01 vs. control. P <0.05 vs. control.

10)

30 mm

hr' ,nl' 60 mm

67 8

9

270

27

531

49

1b

36

5b

97

13"

38

13

85

20

635

98

128

20c

l3

339 62d

398 44

764 71d

149

345 62

SEM. Time 0 mm indicates the end of the 20-mm equilibrium period. N are numbers of experiments.

01

a)

C

0 a)

***

C C

I

a,

z

L. z I

CO

0

.

C

*

.1—

a)

611

______

ft

01

0

•:::••.::::.;:.::. z

-.

0

E

Fractional renin release, %

Sodium balance and renin regulation

4 3 5 (N=7) (N=10) (N9) (N=6) Fig. 2. Renin secretion in vitro: Renin secretion rate and fractional renin release in the isolated perfused rat kidney. Mean values (bars) and 1 SEM (small vertical lines) are shown. Group numbers are indicated under each bar. Small numbers in parentheses are numbers of experiments. Probability values are calculated vs. the value in group I (controls) and indicated by asterisks: * <0.05, *** <0.001; NS 2 4 5 3 Group 1 (N=14) (N=7) (N=1O) (N=9) (N=6)

2

1

(N=14)

= nonsignificant.

Discussion

A

Increasing data indicate that changes in plasma renin levels are not directly proportional to those in renal renin content [7, 8, 22], when the sodium balance is altered in various degrees. Our data in vivo are in agreement with these observations. Whereas the RR varied in a relatively small range, a wide

range of PRC variation was observed both in anesthetized and nonanesthetized rats. Though PRC is not a direct measure of renin secretion rate in vivo, it probably reflects the activity of a reninsecreting mechanism, especially in nonanesthetized

animals. Because acid-activatable renin was not found in the rat plasma samples in any of the groups examined, the PRC differences among these groups

cannot be attributed to the secretion of different amounts of inactive renin, as proposed in humans under certain conditions [12, 23, 24]. The absence

0

1.0

C

00

S 4-

0

ft a)

0

0

100

C

0

a)

U a)

B

2

C

= a,

£

cc

N = 30 = 0.25 A

of acid-activatable renin in rat plasma has been sug-

gested by previous reports [17, 25], although contradictory data are also reported [13, 26]. In our study in vitro, acid-activatable renin was not measured in the perfusate, because we recently demonstrated that such renin is not secreted by perfused rat kidney [17]. Our in vitro data show that different sodium regimens induce significant differences in renin release by perfused kidneys. RSR changes, however, are not proportional to the relative PRC differences among the groups studied. RSR in sodium-deprived groups is only slightly higher than RSR in the control group and does not reflect the great PRC dif-

N = 30 r = 0.78

0

0

L

LA

Do

La

a

A

0

a

A

0

0 0

200

Plasma renin content, ng of AIhr' ml'

Fig. 3. Relationships between renin secretion rate (RSR) and plasma renin concentration (PRC) in d/ferent states of sodium balance. The data from the groups 1, 2, and 3 (control + sodiumloaded groups) are compared in panel A; and the data from the groups 1, 4, and 5 (control + sodium-deprived groups), in panel B. Each symbol represents an individual experiment. Groups are indicated by different symbols. D = group I;. = group 2; 0 = group

3; = group 4; A = group 5. Regression lines and coeffi-

cients of correlation are shown.

612

Nakane et a!

ferences between these groups (Fig. 3b). On the

large, slowly-mobilized pool [29]. The existence of

other hand, the relative difference in RSR between the control and sodium-loaded groups is similar to their PRC differences (Fig. 3a). This discrepancy is not fully explained by the difference in the magnitude of changes in RR between the two opposing states of sodium balance. When compared in terms of FRR, there is a significant difference between the

the slowly mobilized pool recently has been discussed in connection with an inactive form or a

control and sodium-loaded groups, but no difference was found between the control and sodiumdeprived groups; that is, the slight RSR rise in sodium-deprived groups is proportional to their slight

RR increase. The RSR and FRR reflect different levels of activity for renin-release mechanisms intrinsic to each kidney in the absence of extrarenal regulation. Activity changes in such mechanisms may surely contribute to reduce PRC and RSR disproportionately to the RR decrease in sodium-loaded animals. Because FRR values are not different between the control and sodium-deprived groups, the mechanisms that raise PRC disproportionately to the RR increase in sodium-deprived animals are not preserved under in vitro conditions. This relationship between renin secretion in vivo and in vitro during sodium deprivation has not been reported,

although there have been several works showing that sodium balance variation affects the renin release in vitro [7, 22, 27, 28]. Our results imply the importance of renal intrinsic mechanisms for reducing plasma renin levels during sodium loading and

the importance of other mechanisms for raising plasma renin levels during sodium deprivation. Hence, two questions are raised: (1) what are the renal intrinsic mechanisms that reduce the renin mobilization in vitro as well as in vivo? (2) What mechanisms enhance the renin mobilization in vivo in sodium-deprived animals and are not preserved in vitro? Related to the first question, previous data suggest that there might be two types of renin pool in the kidney: a small, readily releasable pool and a

high-molecular-weight form of renin on the basis of biochemical [30, 31] and histologic evidence [8, 11]. Although the renal secretion of inactive renin is unlikely in rats [17], it is possible that the size of such pools, as well as the rate of transfer of renin molecules between them, varies following the changes in sodium balance. On this assumption, the persistent reduction in vitro of renin release in sodium-loaded

kidneys would reflect depletion of the rapidly releasable renin pool and a reduced rate of its repletion. Moreover, the in vitro data from sodium-deprived kidneys may indicate that the size of this particular renin pool does not greatly increase, and that the apparent reduction in renin release as measured in vitro may be due to the absence of certain factors (for example, extrarenal factors) that stimulate the

repletion of the readily releasable pooi, as well as the renin discharge from it. For the second question, the particularity of the

in vitro conditions must be considered in connection with the factors known to affect renin release. In vitro perfusion eliminates the influences of extrarenal factors, such as neural, humoral, and hemodynamic factors, or renin release under different states of sodium balance. Among these factors, the importance of neural factors in renin control during

sodium deprivation has been stressed by several workers [32, 33]. The assumption that high plasma renin levels during sodium deprivation are maintained principally by extrarenal factors can conve-

niently explain the in vitro results of the present study. Besides, in vitro perfusion may also modify the functional state of certain intrarenal feedback systems involved in renin release regulation. For example, the effects on renin release of the prostaglandin systems [34, 35], of the kallikrein-kinin systems [36], and of the renin-angiotensin system itself

Table 2. Functional characteristics of isolated perfused rat kidneysa

Na

Perfusion

pressure mm Hg

Groups (1) Control (N 14) (2) Sodiumloading + DOCA(N = 9) (3) Sodium loading (N = 7) (4) Sodium deprivation (N = 6) (5) Sodium deprivation + furosemi de(N

71

73 71 68

= 10)

70

I I 1 1

1

Perfusate flow rate ml . min 22 23 23 22 23

GFR

ml 'min'

1

0.40 0.54 0.46

1

0.53

1

0.46

1 1

Urine volume

jd mm1

0.04 0.07 0.06 0.04 0.05

a Values are the means SEM calculated for all 1-hr experiments. N denotes numbers of experiments. h p
P <0.01 vs. control. P < 0.05 vs. control.

37 45 48 65 74

6 9 7

I0' 6b

reabsorption %

92 93 91 91 84

I I I 2 2C

Sodium balance and renin regulation

pression by DOC and NaCl in the rat. Am J Physiol

[37] may be altered in vitro because of the lack of

plasma substrates. Our results can also be explained by assuming that the functional modification of such intrarenal systems under in vitro conditions is a crucial disadvantage for sodium-deprived kidneys to maintain an enhanced renin release. In

fact, sodium-deprived kidneys showed slightly lower tubular capacity to retain sodium compared with kidneys in other groups, which is probably not

the case in vivo. This paradoxical observation might be related to the acute elimination in vitro of extrarenal supports for the kidney to retain sodium [38, 39]. The in vitro perfusion also may inhibit the

sodium-deprived kidney from secreting a large quantity of renin, through such functional changes. The inability of sodium-deprived kidneys to main-

tain an enhanced renin release in vitro contrasts with the persistent reduction of renin release in vitro from sodium-loaded kidneys.

613

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DAVIS JO, FREEMAN RH: Mechanisms regulating renin re-

lease.Physiol Rev 56:1-56, 1976 11. DE SENARCLENS CF, PRICAM CE, BANICHAHI FD, VALLOTToN MB: Renin synthesis, storage, and release in the rat: A

morphological and biochemical study. Kidney mt 11:161169, 1977 12. SEALEY JE, MooN C, LARAGH JH, ATLAS SA: Plasma pro-

renin in effect

45,

normal, hypertensive, and anephric subjects and its

on renin measurements. Circ Res 40 (suppl 1):I-41—I-

1977

13. VANDONGEN R, POESSEE M, STRANG KD, BIRKENHAGER

WH: Evidence that 'inactive renin' is produced outside the kidney of the rat. Clin Sci Mol Med 53:189-191, 1977 14. BOWMAN RH, MAACK T: Glucose transport by the isolated perfused rat kidney. Am J Physiol 222:1499-1504, 1972 15. NAKANE H, NAKANE Y, REACH G, CORVOL P, MENARD J:

Aldosterone metabolism in isolated perfused rat kidney. Am 1978 16. MENARD J, CATT KJ: Measurement of renin activity, conJ Physiol 234:E472-E479,

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Acknowledgment

A portion of this work was presented at the 8th World Congress of Cardiology, Tokyo, in September, 1978. This work was supported by grants from INSERM, DGRST, and l'Association Claude Bernard. Reprint requests to Dr. Haruyuki Nakane, INSERM U36, 17, rue du Fer-à-Moulin, 75005 Paris, France

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Iowa State University Press, 1967 21. CARVALHO iS, SHAPIRO R, HOOPER P, PAGE LB: Methods

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