AMERICAN
JOURNAL
OF
Vol. 228, No. 3, March
Plasma
PHYSIOLOGY
1975.
in U.S.A.
Printed
renin
activity
during
ovine
pregnancy
ALAN R. FLEISCHMAN, GARY K. OAKES, MICHAEL F. EPSTEIN, KEVIN J. CATT, AND RONALD A. CHEZ Pregnancy Research Branch and Section on Hormonal Regulation, Ke/woduction Research Branch, Nutional Child Health and Human I)evelopmen t, Xational Institutes of Health, Bethesdu, Murylund 20014
FLEISCHMAN, ALAN Ii., GARY K. OAKES, MICHAEL F. EPSTEIN, KEVIN J. CATT, AND RONALD A. CHEZ. Plasma renin activity during ovine pregnancy. Am. J. Physiol. 228(3): 901-904. 19XChronic intravascular catheterization in maternal, fetal, and neonatal sheep was utilized to assess basal plasma renin activity (PRA) and changes in PKA in response to furosemide. Maternal PRA increased from base-line levels during the last trimester of pregnancy and remained elevated for 12 wk postpartum. Fetal basal levels of PRA were variable but usually greater than maternal levels. Intravenous administration of furosemide to pregnant ewes resulted in a prompt and significant increase in maternal YRA with inconsistent changes in fetal PRA. Fetal and newborn responses to the direct intravascular injection of furosemide were apparently dependent on the basal level of PRA. Fetal and neonatal animals with low basal levels showed a significant increase in PRA; maternal PRA did not change. Animals with higher basal levels did not respond to the stimulus, perhaps reflecting a maximum renin secretory rate. These data are consistent with the conclusions that fetal renin originates predominantly from the fetal kidney, that fetal PRA receives no significant contribution from the maternal circulation, and that renin does not cross the ovine placenta. renin-angiotensin
system;
fetal
physiology;
salt
and
water
me-
tabolism
THE RELATIONSHIP BETWEEN the changes in maternal and fetal salt and water metabolism and changes in the reninangiotensin-aldosterone system during gestation is not clear. In normal human pregnancy, maternal Ievels of plasma (PRC), renin activity (PRA), pl asma renin concentration and plasma renin substrate (PRS) are greater than nonpregnant levels (2, 8, 11, 22, 28). Granulated juxtaglomerular cells are present in the human fetal kidney at 17 wk gestation (17), and human uxnbilical cord plaslna renin levels have been measured in the 3rd trimester of pregnancy (2, 9, 14, 16, 22, 25, 26). However, the time in fetal life when renin secretion begins and the function of fetal renin are not known. The pregnant sheep has been used in several studies to assess perinatal changes in the renin-angiotensin systenl. In acute experiments performed under anesthesia, fetal PRA and PRC increased in response to administration of a diuretic to the fetus, after fetal aortic constriction, and with acute fetal blood loss (20, 23, 29). However, anesthesia, stress, and surgical manipulation have been shown to modify PRA and PRC (19, 2 1). To avoid these variables, the present study was performed upon chronically cathc-
Institute
of
terized pregnant sheep to determine basal PRA during the perinatal period, and to evaluate the changes in maternal, fetal, and neonatal PRA in response to a defined pharmacologic stimulus. METHODS
Biweekly blood specimens from five pregnant Dorset ewes with accurately timed gestations were obtained from 64 days until term (145 =t 5 days). Sampling was continued from these mothers and their newborn lambs for up to 4 mo postpartum. Blood samples were also obtained from 15 nonpregnant Dorset ewes for determination of PRA. All sanlples were taken at 0800 h from the jugular vein of quietly standing animals prior to feeding. Animals were provided with standard pellet diet (Wayne Feed Supply Co., Inc., Gaithersburg, Md. Na, 130 meq/kg food. K, 259 meq,/kg food) and water ad libitum, with no medication or salt restriction. Additional pregnant Dorset ewes were utilized for chronic maternal and fetal intravascular catheterization in the 3rd trimester. Fetal dorsal pedal artery and vein catheters as well as maternal saphenous artery and vein catheters were placed by the technique of Battaglia (1). Specifically, after a 48-h fast, a 3-cm hysterotomy was performed under spinal or intravenous ketamine anesthesia, and polyvinyl catheters were placed in the fetal hindlimb. Fetal membranes, uterine wall, and abdominal musculature were closed and the catheters were tunneled subcutaneously to exit through a O-5-cm incision in the maternal left flank, where they were placed with the maternal catheters in a cloth pouch. Postoperatively, animals were housed in specially constructed metabolic cages. Recovery to the point of being able to stand and eat occurred within 4 h of the surgery. All animals received 600,000 U procaine penicillin and 0.5 g streptomycin sulfate intramuscularly on the day of surgery and for the 3 days following. Animals were allowed free access to food and water except during an experiment. Daily blood sampling, as well as acute experiments, were performed at 0800 h, with the animal standing. Both were initiated on the 4th postoperative day and with at least a 3-day interval between experiments. The status of the fetus was assessed by periodic hematocrit, blood pH, and blood gas analyses. Furosemide (Hoechst Pharmaceutical Co., Sommerville, a potent diuretic in sheep (6), was given intraN- .I*>, venously in a dose of O-5-0.6 mg/kg to five mothers at 130-137 days’ gestation. Blood samples (1.2 ml) were ob-
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902 tained from fetus and mother sequentially over 2 h. A total volume of 8.5 ml was removed from each mother and fetus within the 2-h period studied, and was replaced with an equal volume of normal saline. In separate experiments, 3 or 6 mg of furosemide were given intravenously directly to nine fetuses of gestational age 128-136 days, and similar sampling was carried out- Six different, newborn animals, allowed to deliver spontaneously at term and kept with their mothers until the experiment, were given 3 mg of furosemide intravenously on the lst, Znd, or 3rd day of life vice an external jugular catheter. Again, blood samples were obtained over a Z-h period. In five control preparations, sequential blood samples were obtained from mother and fetus over a 2-h period without administration of the drug. In other animals, catheters were placed in the urinary bladders of three mothers and two fetuses to measure the renal response to furosemide administration. The animals with chronically implanted intravascular catheters were not subjected to this surgical manipulation. Blood samples were placed into Vacutainer tubes containing EDTA and kept on ice until centrifuged at 4°C for 15 min at 1,500 X g. Plasma samples were stored frozen at - 15OC until assayed for PRA by a minor modification of the method previously described for determination of PRA in human plasma (4). Sheep plasma samples were incubated at 37°C for 3 h, after dilution 1: 1 with 0.2 M phosphate buf’ler, pH 7.0. Each sample contained 5 mM disodium EDTA, 2.5 m&I 8-hydroxyquinoline sulfate, and 5 mM BAL (2,3-dimercapto1-propanol). An aliquot of each sample was kept in ice for 3 h and subjected to radioimmunoassay for angiotensin I with the incubated sample to give the “4OC blank,” which was subtracted from the value for angiotensin I generation in the incubated sample. Radioimmunoassay for angiotensin I was performed as previously described (4), and results were expressed as nanograms per miililiter plasma per hour in terms of a synthetic standard of [ Ile”]angiotensin I (Schwarz/Mann), which had been calibrated according to the amino acid analysis and UV absorption (275 nrn) of a standard solution of the native peptide. The within-assay precision of the assay for PRA at the levels measured in sheep plasma was + 7 c/D ( toe i1‘lcient of variation), and the between-assay precision was + 15 % .Results were analyzed utilizing nonpaired t-test statistics. RESULTS
The mean (+SD) level of PRA in 15 nonpregnant control animals (0.43 & 0.26 ng,/ml per h) is compared in Fig. 1 with the values obtained throughout gestation and the postpartum period in five nonoperated pregnant sheep. PRA increased during the last trimester of pregnancy and remained elevated in the postpartum period. A return to nonpregnant levels had occurred by 3 mo after delivery. Figure 2 records the daily (mean & SE) maternal and fetal PRA in 15 chronically catheterized animals during late gestation. The fetal: maternal ratio from individual animals was usually greater than 1. Maternal values for PRA from 128 to 140 days were relatively constant, with means ranging from 2.2 to 4.0 ng/rnl per h. Simultaneous fetal basal values during this time period were more vari-
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FIG. 1. Sequential maternal PR,\ throughout ovine gestation in postpartum period (n = 5). Toned area represents mean (n = 15) for nonpregnant ewes. Solid line is best-fit regression nomial from daily mean values throughout gestation,
180 T
I
T I MEAN Z!I
I I
SEM
9,:
+
and SD poly-
T Fetus Mother (N= 15)
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128
130
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132
134
GESTATIONAL
2. Fetal and maternal PR,4 chronic intravascular catheters.
FIG.
with
I
136 AGE
during Term
138
140
(day:)
late gestation
gestation is 145
in sheep =t 5 days.
able, with daily means ranging from 6.6 to 15.5 ngjml per h. Sequential samples from individual healthy fetuses showed marked daily variation ranging from 0.3 to 25.0 ng,/ml per h in late gestation. Newborn PRA was found to be consistently greater than puerperal maternal levels and remained elevated, although they decreased toward adult nonpregnant levels by the end of the 1st mo of life. Values ranged from 3.6 to 25.5 ng/rnl per h in the 1st wk of life to 0.4 to 8.0 ng/xnl per h by the 4th wk of life. The intravenous administration of furosemide to five pregnant ewes was followed by a prompt and significant increase in maternal PRA after 15 min, rising at 30 min to a peak value 2 lo-340 $S of basal levels (Fig. 3). There was no consistent change in fetal PRA in these experiments, although there was a trend toward a late rise in fetal PRA at 90-120 min.
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PERINATAL
PLASMA
RENIN
903
ACTIVITY
These data can be contrasted with the effect of furosemide injected directly into the near-term fetus. There appeared to be two fetal groups, responders and nonresponders, with no difference exhibited relative to the dose of 3 or 6 mg of furosemide. Figure 4 depicts the results of administration of 3 mg furosemide to two representative animals. Three fetuses with basal PRA greater than 8.0 rig/ml per h did not show increased PRA following the injection of the diuretic, whereas six fetuses with lower basal PRA responded promptly and significantly with an increase to 320-l 780 % of the basal levels. There was no change in maternal PRA in any of the fetal experiments. Newborn lambs could similarly be separated into responsive and nonresponsive groups on the basis of the changes in PRA levels following the infusion of diuretic. Basal PRA levels were signiiicantly higher (range 11.641.1 ng/mI per h) than those observed in the fetus. Two of the six newborns had basal PRA levels less than 16.0 rig/ml per h, and responded with an increase in PRA to 2 17 and 237 % of basal values. These two animals were studied on the 1st day of life. Four other animals on the 2nd and 3rd days of life, with basal PRA of 27.0 to 43.0 rig/ml per h, did not respond to the diuretic with an increase in PRA. In the control animals, sequential blood sampling over 2 h from mother and fetus resulted in no change in maternal PRA. Fetal PRA values remained stable for the Grst 60 I8.0 n
Fetus
M
15.0
MEAN
Mother f SEM
(N = 5)
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9.0 6.0 T
3.0 0
FIG.
I I -10 0 t InjectIon
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TIME
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of furosemide
I +30
(0.5-0.6
and fetal mg/kg
M
PRA body
Fetus Mother
>’
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I
I
+60
+90
+120
(minutes)
response to intravenous wt) to mother.
4,2
w-4 k-4
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Fetus Mother
I 0
I
I
+I5
+30
f
Injectron FIG. 4, Maternal and fetal PRA of furosemide (3 mg) to 2 fetuses.
483
-- ----
---------v -I-
I -10
I
+60 TIME
injection
I --I
+90
-4 -- -4,t 1
+120
(minutes)
response
to intravenous
injection
min, and appeared increased, although not significantly, above basal levels at 90-l 20 min. This was comparable to the change seen in fetal PRA during furosemide administration to the mother. In the three mothers in which the bladder was catheterized, the urine production rate increased eightfold over basal levels in the 30 min after the intravenous injection of furosemide. Diuretic administration directly to two fetuses resulted in a fivefold increase in fetal urine volume from base line during the subsequent 30 min. DISCUSSION
Marked increases in maternal PRA, PRC, and PRS have been observed during human gestation (2, 8, 11, 22, 28). In humans, maternal and fetal levels of PRS are elevated during pregnancy by an increase in hepatic synthesis secondary to the high levels of circulating estrogens (10, 3 l)* When PRS is elevated, PRA becomes relatively independent of PRS levels and is more directly proportional to enzyme concentration (5). During sheep pregnancy, maternal PRA and PRC have been reported to be elevated above the nonpregnant levels (20, 23, 29). The present study adds longitudinal evidence concerning the rise in maternal PRA throughout ovine gestation and in the postpartum period. In this study, sheep fetal P.RA was found to be extremely variable in Iate gestation, but was consistently greater than the maternal levels. That is, the fetal maternal ratio was usually greater than 1. There has been disagreement concerning the fetal : maternal ratio of PRA and PRC in humans (9, 14, 16, 20, 22, 3 1) as well as in sheep (20, 23, 29). This inconsistency may be due to the changes in maternal, and perhaps fetal, renin values induced by labor, anesthesia, surgical manipulation, and delivery (8, 25). F or example, fetal: maternal ratios of PRC obtained from acute surgical preparations in sheep were consistently less than 1 (29), whereas ratios obtained in the same laboratory in chronically catheterized animals were uniformly greater than 1 (20). Term-newborn human plasma renin levels are greater than adult values for at least 6 wk after delivery (9, 26). In like manner, the PRA of term-newborn sheep in the present study was consistently elevated above adult levels during the 1st mo of life. This may be causally related to or result from the decreased renal function of the neonate (16). Furosemide is a potent stimulus to renin secretion in many species (18, 30). T rimper and Lumbers (29) demonstrated in acutelv anesthetized supine sheep that four of five Dregnant ewes and all four n&pregnaAt ewes did not sh;w-a significant increase in PRC in response to intravenous furosemide (0.5 “g/kg). In the present study utilizing standing, chronically catheterized, unanesthetized animals, a prompt increase in maternal PRA consistently followed the intravascular injection of furosemide (0.5-0.6 “g/kg). By contrast, fetal changes in PRA after maternal injection of diuretic were not consistent. There appears to be a late rise in fetal PRA in control and maternally injected animals, perhaps reflecting fetal blood loss or acute maternal-placental shifts in water and salt balance. It is not known whether furosemide crosses the sheep placenta- Because the drug is strongly bound to plasma protein, it is probable that it does not (29). The fetal and newborn responses to direct intravenous
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 13, 2019.
904 injection of furosemide appeared to depend on the basal level of PRA. The higher basal levels may reflect the maximum renin secretion rate which can be achieved by the fetal and early neonatal kidney; additional stimulation in such animals would evoke no further increase in release of the enzyme. The role of fetal renin, and the physiological conditions under which it can be modified, have not yet been clarified. The contribution of utero-placental-fetal renin to maternal renin levels has been studied in several species (3, 7, 8, 15, 24). It has been suggested that renin freely crosses the canine placenta ( 13). However, nephrectomized pregnant dogs show no change in PRA in response to multiple stimuli which increase PRA in nonnephrectomized mothers (3). Similar studies in rabbits have demonstrated an increase in PRA in the nephrectomized mothers (7). Such discrepancies have also been observed in the human being. Simultaneously obtained human uterine vein and peripheral vein PRA and PRC at the time of elective cesarean section delivery have demonstrated higher levels at both sites (15, 22, 24). Maternal and fetal renin levels in the absence of fetal kidneys have been investigated in several species (12, 20, 26). Fetal nephrectomy in monkeys was followed by variable changes in maternal levels of plasma renin (12). Pre-
FLEISCHMAN
ET
AL.
liminary data from studies utilizing chronically nephrectomized sheep fetuses show a profound decrease in fetal PRA and PRC with no change in maternal levels ((20) and unpublished observations). This is consistent with data obtained from a term human anephric fetus in which umbilical cord PRA was undetectable, PRC was less than 10 %, of normal cord values, and the PRS level was within the normal range (26). It can be concluded from these reports and the present study that human and ovine fetal renin originates predominantly from the fetal kidney, and receives no significant contribution from the maternal circulation. Specifically, significant transport of renin across the ovine or primate placenta ap.pears unlikely. Although not directly affecting maternal renin levels, the variability of basal fetal PRA and its response to a pharmacological stimulus are consistent with the possibility that renin has a function in the fetus. This may be related to regulation of uterine blood flow, fetal renal function, and sodium metabolism, or maintenance of fetal blood pressure. The chronically catheterized pregnant sheep provides an appropriate animal model with which to study this COIZIplex system. Received
for
publication
19 July
1974.
REFERENCES 1. BATTAGLTA, F. C. Placental clearance and fetal oxygenation. Pediatrics 45 : 563-575, 1970. J. J., D. L. DAVIES, P. B. DOAK, A. F. LEVER, AND J. I. J. 2. BROWN, ROBERTSON. Serial estimation of plasma renin concentration during pregnancy and after parturition. J. Etxdocrinol. 35: 373-378, 1966. I3 . CARRETERO, 0. A., C. POT,ANSKI, A. PXWONSKI, A. AT;SARI, AND C. P. HODGKINSON. Renin release and the uteroplacental-fetal complex. Am. J. physiol. 223 : 56 l-564, 1972. 4. CATT, K. J., A. J. BAUKAL, AND M. J. ASHBURN. In: Oral Conkception and High Blood Pressure, edited by M. J- Fregly and Al. S. Fregly. Gainesville, Fla . : Dolphin, 1974, p. 184-2 10. 5I . CA’I’T, K. J., A, J. BAUKAL, AND M. J- ASHBURN. In: Oral Contracefition and High Blocd Pressure, edited by M. J. Fregly and M. S. Fregly. Gainesville, Fla. : Dolphin, 1974, p. 21 l-226. 6. ENGX.ISH, P. B., AND W. E. BECVAR. Effects of furosemide on the external balances of water, sodium, potassium and chloride in sheep. Am. J. Vet. Res. 32: 1371-1379, 1971. 7. FERRIS, T. F., J. H. STEIN, AND J. KAUFFMAN. Uterine blood flow and uterine renin secretion. J. c!in. Invest. 51 : 2827-2833, 1972. 8. GEELHOED, G., AND A. VANDER. Plasma renin activity during pregnancy and parturition. J. C/in. Endocrinol. Metab. 28 : 412415, 1968. 9. HAYDUK, K., D. K. KRAUSE, R. HUENGES, AND V. UNBEE-IAUN. Plasma renin concentration at delivery and during the newborn period in humans. Exfierientia 28 : 1489-1490, 1972. 0. M., AND R. S. GR~FFIY-H. The effect of the adminis10. HELMER, tration of estrogen on the renin-substrate (hypertensinogen) content of rat plasma. Endocrinology 5 1 : 42 l-426, 1952. 0. M., AND W. JUDSON. Influence of high renin sub11. HELMER, strate levels on renin-angiotensin system in pregnancy. Am. J. O&et. Gynecol. 99 : 9-l 7, 1967. 12. HODARI, A. A. In : Physiological Biochemistry of the Fetus, edited by A. A. Hodari, and F. Maniona. Springfield, Ill.: Thomas, 1972, 256-276. 1.3. HODGKINSON, C. P., A. A. HODARI, AND F. M. BUMPUS. Experimental hypertensive disease of pregnancy. Obstel. Gy Izecol. 30 : 371-380, 1967. 14. KU-Z, F., P. BECK, AND E. MAK~WSK~. The renin-aldosterone system in mother and fetus at term. Am. J. Obstet. GynecoE. 118 : 51-55, 1974. 15. KOKOT, F., AND A. CEKANSKI. Plasma renin activity in peripheral and uterine renin blood in pregnant and nonpregnant women. J. O&et. Gynaecol. Bit. Commonwealth 79 : 72-76, 1972.
16.
KOXHEN, T. A., A. L. S’TRICKI.AND, T. W. RICE, AND D. R. WAL’I’ERS. A study of the renin-angiotensin system in newborn infants. J. Pediat. 80: 938-946, 1972. 17. LJUNGQVIST, A., AND J. WAGERMARK. Renal juxtaglomerular granulation in the human foetus and infant. Acta Pa&Z. Micl-obiol. &and. 67 : 257-266, 1966. 18. MEYER, P., J. ~,~ENARD, N. PAPANICOLAOU, J. M. ALEXANDER, C. DEVAUX, AND P. MILLIEZ. Mechanism of renin release following furosemide diuresis in rabbit. Am. J. Physiol. 2 15 : 908-9 15, 1968. 19. PETY-INGER, W. A., M. MARCHET.LE, AND L. AUGUSTO. Renin suppression by DOC and NaCl in the rat. Am. J. Physiol. 221 : 10711074, 1971. F. B., E. LUMBERS, AND J. MOTT. Plasma renin and angio20. PIPKIN, tensin II in conscious pregnant ewes and their lambs. J. physiol., London 237: 52-53, 1973. D., AND A. h/C. MICI~-XEI,AKIS. Effect of anesthesia and 21. I