Circadian Changes in Plasma Renin Activity and Plasma Aldosterone Concentration in Two-Kidney Hypertensive Rats

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1 Circadian Changes in Plasma Renin Activity and Plasma Aldsterne Cncentratin in Tw-Kidney Hypertensive Rats JACK M. DEFORREST, PH.D., JAMES O. DAVIS, PH.D., M.D., RONALD H. FREEMAN, PH.D., GREGORY A. STEPHENS, PH.D., AND BARRY E. WATKINS, PH.D. with the technical assistance f Charles H. Gay SUMMARY Circadian changes in plasma renin activity (PRA) and plasma aldsterne cncentratin (PAC) in nrmal and hypertensive rats were determined by measurements at 8 a.m., 4 p.m. and 12 midnight (MN). Fr the nrmals, PRA and PAC were highest at 4 p.m. Animals made hypertensive by cnstricting ne renal artery with the ther kidney intact were studied after 4,5,7 and 10 weeks; the clear-cut circadian rhythm fr PRA in nrmals had disappeared but fr PAC the circadian rhythm was present in the 4-, 5- and 10-week grups. Bth PRA and PAC were elevated in all fur hypertensive grups cmpared with the nrmal cntrls and there was a highly significant crrelatin between PRA and PAC. The 4 p.m. peak value fr PAC was much higher in relatin t the 8 a.m. and 12 MN values in the hypertensive animals than in the nrmals. Sdium balance studies failed t demnstrate any appreciable differences amng the grups. When the hypertensive animals were divided int tw grups n the basis f the level f hypertensin, the rats with mderate hypertensin shwed an average elevatin in PRA which was significant in nly the 4- and 7-week grups whereas PRA was elevated in all fur grups with severe hypertensin. Thus, the present data help t define the activity f the renin-angitensin-aldsterne system in tw-kidney, ne clip hypertensin in the rat. (Hypertensin 1: ,1979) KEY WORDS renin-angitensin-aldsterne system hypertensin aldsterne secretin circadian rhythm f plasma renin activity experimental renin hypertensin renin secretin high bld pressure BEFORE the rle f the renin-angitensinaldsterne system can be adequately evaluated in tw-kidney, ne clip hypertensin in the rat, any circadian rhythm f this hrmnal system must be knwn. Leenen et al. 1 and Gmez-Sanchez et al. a have previusly described circadian changes fr bth renin and aldsterne in the nrmal rat, but n cmparable studies have been reprted fr the renal hypertensive rat. The findings frm studies f the rle f the reninangitensin-aldsterne system in the tw-kidney hypertensive rat have nt always agreed. Pssible explanatins fr these variatins include the methds f bld sampling, whether the animals were anes- Frm Department f Physilgy, University f Missuri Schl f Medicine, Clumbia, Missuri Address fr reprints: Dr. James O. Davis, Department f Physilgy, University f Missuri, Schl f Medicine, Clumbia, Missuri thetized r cnscius, and, as has been recently demnstrated, the severity f hypertensin.' Anther pssible explanatin might be variatins in circadian changes in the plasma levels f renin and aldsterne in the hypertensive rat. The purpse f this study was threefld: 1) t redefine the circadian changes in plasma renin activity (PRA) and plasma aldsterne cncentratin in nrmal rats; 2) t examine any changes in the circadian pattern fr PRA and plasma aldsterne cncentratin in the tw-kidney hypertensive rat; and 3) t determine if PRA and the plasma aldsterne level return t nrmal in hypertensive rats studied fr 10 weeks. Methds All rats (Sprague-Dawley) were hused individually in stainless steel metablic cages and kept in temperature-cntrlled rms that were maintained n a 12-hur light-dark cycle beginning at 5 a.m.

2 CIRCADIAN CHANGES IN HYPERTENSION/Z>eFrr«r et al. 143 Distilled water and pwdered rat fd (Purina) were available ad libitum. Sdium balances were measured befre sacrifice frm tri-weekly bservatins f ingested sdium and sdium excreted by the kidney; three 2-day sdium balance perids were btained. Systlic bld pressure was determined by the tail artery cclusin methd; the value recrded was the average f three determinatins which were made twice weekly. Plasma renin activity was determined frm bld cllected during the first 5 secnds fllwing decapitatin, while plasma aldsterne cncentratin (PAC) was measured frm a secnd bld sample cllected during the next 15 secnds. Bld samples (5 ml) were cllected in 0,1 ml f 10% ethylenediaminetetraacetate; the plasma was frzen until subsequent analyses were made by radiimmunassay. Fr plasma sdium and ptassium cncentratins, 1 ml f bld was cllected during ether anesthesia frm nrmal, as well as frm 4-, 5-, 7- and 10-week hypertensive rats by cardiac puncture at each f the 8 a.m., 4 p.m. and 12 midnight (MN) times. Fllwing recvery frm the anesthesia, the nrmal animals were decapitated and bld again cllected s that plasma sdium and ptassium cncentratins by this cllectin prcedure culd be cmpared with bld cllected by cardiac puncture. Hvpertensfre Studies Eight separate grups with male rats in each grup were used in this study. Systlic pressure was measured twice prir t placing a 0.2-mm silver clip n the left renal artery; the bdy weight was g. The cntralateral kidney was left untuched. After 4 weeks bld pressure was determined n tw f the grups; these grups were then randmly subdivided int three smaller grups f 5-6 rats each fr subsequent sacrifice fr cllectin f bld at 8 a.m., 4 p.m. r 12 MN. This prtcl was als fllwed at the end f Weeks 5, 7 and 10 fr the remaining grups f rats. Sham Cntrl Studies Fur grups f 18 nrmal male rats per grup were studied after 4 weeks (average bdy weight = 250 g). Three grups were examined during a 2-mnth perid (May-June), while the furth grup was studied several mnths later (January). Systlic bld pressure was determined during the week befre sacrifice. As with the hypertensin studies, each cntrl grup was then subdivided int three grups s that equal numbers culd be sacrificed t btain bld at 8 a.m., 4 p.m. r 12 MN. Analytical Techniques and Statistics The radiimmunassay techniques f Sealey et al. 4 and Buhler et al. 5 were utilized fr analyzing PRA and plasma aldsterne cncentratin, respectively. Urine and plasma electrlytes were measured by flame phtmetry. Statistical significance was determined between grups by Student's grup r unpaired t test. Results Systlic bld pressure was 106 ± 1.4 SEM mm Hg fr the sham perated cntrl grup (fig. 1). Placing a 0.2-mm clip n the left renal artery increased bld pressure frm pre-clip cntrl values f 107 t 188 mm Hg, frm 104 t 180 mm Hg, frm 116 t 210 mm Hg, and frm 107 t 192 mm Hg, fr the 4-, 5-, 7- and 10-week hypertensive grups, respectively (fig. 1) (p < 0.01 fr all fur grups). N significant differences were bserved in the mean values fr pressures recrded n rats sacrificed at 8 a.m., 4 p.m. and 12 MN within any grup (p > 0.05). The death rate f each grup due t factrs ther than anesthesia r surgery is als presented; mre rats died in the 10- week hypertensive grup than in any ther grup. Nne f the cntrl rats died during the study. During the week befre sacrifice, the average value fr the sdium excreted by the kidney was apprximately 82% f the ingested sdium fr all rats studied (fig. 2). The rati f excreted t ingested sdium was quite variable within each grup but there were n bvius differences in sdium balance between any f the experimental grups and the nrmal cntrls. Of all the hypertensive animals, nly five demnstrated a negative sdium balance; these rats were prbably in the malignant phase f hypertensin. Thesefiveanimals shwed a mean systlic pressure f 228 ± 5 mm Hg, a PRA f 7.1 ± 1.1 ng angitensin I/ml/hr, and a mean plasma aldsterne cncentratin f 86 ± 26 ng%. These values fr arterial pressure, PRA and plasma aldsterne cncentratin are very high cmpared with thse recrded in nrmal rats (figs. 1-3). Plasma renin activity and plasma aldsterne cncentratin exhibited a reprducible circadian change in the nrmal rats. As seen in figure 3, PRA f rats sacrificed during May-June, 1977 was significantly higher at 4 p.m. than at 12 midnight (p < 0.05). Plasma aldsterne cncentratin in the same rats fllwed a similar rhythm; the 4 p.m. values were significantly higher than either the 8 a.m. r 12 midnight values (p < 0.05 fr bth cmparisns). Several mnths later (January, 1978), this experiment was repeated with similar results; PRA was significantly higher at 4 p.m. than at 8 a.m. r 12 MN. Plasma aldsterne cncentratin als appeared t be higher at 4 p.m. than 8 a.m. r 12 MN but the differences were nt statistically significant. Data frm these tw grups were added tgether fr bth renin and aldsterne and subsequently referred t as data frm the cntrl grup. Within this cmbined grup (figs. 4, 5), PRA rse significantly frm 0.72 ± 0.06 ng angitensin I/ml/hr at 8 a.m. t 0.97 ± 0.01 ng angitensin I/ml/hr at 4 p.m., and then fell t 0.58 ± 0.06 ng angitensin I/ml/hr by midnight (p < 0.05 fr bth cmparisns). The cncentratin f plasma aldsterne at 4 p.m. was 16.1 ± 1.8 ng% cmpared with 11.6 ± 1.4 ng% at 8 a.m. and 11.4 ± 1.4 ng% at midnight (p < 0.05 fr bth cmparisns).

3 144 HYPERTENSION VOL 1, N 2, MARCH-APRIL 1979 DEATH RATE % I ~ 225 LJ % 210 jg 195 = 8AM A=4PM = 12 MN 8% 9% 6% A A 21% Q- 180 O Op O t (PA* * * : A CONTROLS C HT C HT C HT C HT 4 WEEKS 5 WEEKS 7 WEEKS 10 WEEKS FIGURE 1. Systlic arterial pressure measurements in nrmal cntrls and hypertensive (HT) rats. Determinatins were made at 8 a.m., 4 p.m. and 12 midnight. Each grup f hypertensives had measurements made befre and at 4, 5, 7 and 10 weeks after renal artery cnstrictin. NA INTAKE CONTROLS 4 WEEKS HT 5 WEEKS HT 7 WEEKS HT 10 WEEKS HT =8 AM A...A=4PM = I2MN DAYS FIGURE 2. Daily sdium balances measured n the furth, the secnd and zer days befre decapitatin. E N, is the abbreviatin fr daily sdium excretin. The symbls fr the 8 a.m., 4 p.m., and 12 midnight grups are fr animals sacrificed at these times fr bld cllectin and each animal had three 2-day balance perids studied.

4 CIRCADIAN CHANGES IN HYPERTENSION/Z?eF/r«f et al L * = 4RM.>l2MNP<05 *=4PM.>8AMP<05 N = I7 -^SACRIFICED JAM, 1978 N = 6 SACRIRCED JUNE, 1977 N \, 7 8 AM 4 PM 12 MN SACRIRCED JUNE, 1977 N=I7 N = I7.SACRIFICED JAM, 1978 N = 6 8 AM 4 PM 12 MN FIGURE 3. Circadian changes in plasma renin activity and plasma aldsterne cncentratin f nrmal rats. The animals were n a 12-hur light-dark cycle. Plasma renin activity f the hypertensive grups was nt significantly elevated at 4 p.m. in cmparisn with the 8 a.m. and 12 MN values (fig. 4). The nly statistical difference within a grup existed in the 7- week hypertensive animals in which PRA was significantly greater at 8 a.m. than at midnight (p < 0.05). Except fr the 8 a.m. value f the 4-week grup, PRA f all hypertensive grups was significantly greater than in the cntrls (p < 0.05). Als, the 8 a.m. value fr PRA at 7 weeks was significantly higher than the PRA values at 4 and 5 weeks (p < 0.05 fr bth cmparisns). As presented in figure 4, PRA appears t increase prgressively frm the 4-week hypertensive grup thrugh the 7- week hypertensive grup. The PRA f the 10-week hypertensive grup appears t be lwer than that f the 7-week hypertensive grup, suggesting that PRA was returning tward the cntrl level. In cntrast, plasma aldsterne cncentratin appears t have a circadian change within three f the fur hypertensive grups (fig. 5). In the 4-week hypertensive animals, the plasma aldsterne level was 16.0 ± 1.0 ng% at 8 a.m. and 63.2 ± 27 ng% at 4 p.m. (p > 0.05), but fell t 13.9 ± 3.0 ng% by midnight (p < 0.05). The 5-week hypertensive animals fllwed a similar pattern as the level f plasma aldsterne was 19.5 ± 5.5 ng% at 8 a.m. and 42.8 ± 10.8 ng% at 4 p.m. (p < 0.05), and then fell t 7 WEEKS HT_ 10 WEEKS HT, * >I2 MN P<O5 >8 AM P<D5 O >CONTROLS P<05 8 AM 4 PM 12 MN FIGURE 4. Circadian changes in plasma renin activity fr nrmal cntrls and fr , 7- and 10-week hypertensive (HT) rats.

5 146 HYPERTENSION VOL 1, N 2, MARCH-APRIL WEEKS HT \ \ *>I2MNP<.O5 / \ * > 8 AM P<05 \\ > CONTROLS P< 05,/V WEKS -CONTROLS 8 AM 4 PM 12 MN FIGURE 5. Circadian changes in plasma aldsterne cncentratin fr nrmal cntrls and fr 4-, 5-, 7- and 10-week hypertensive (HT) rats ± 5.5 ng% by midnight (p > 0.05). The cncentratin f aldsterne in plasma fr the 10-week hypertensive grup was 54.0 ± 28.5 ng% at 8 a.m. cmpared with 90 ± 58 ng% at 4 p.m. {p < 0.05); aldsterne fell significantly at 12 MN t a value indistinguishable frm the cntrl (p > 0.05). Whereas the plasma aldsterne level in the hypertensive rats was significantly higher than cntrl at all times during Weeks 5 and 7, nly the 4 p.m. values f the 4- week grup, and the 8 a.m. and 4 p.m. values f the 10-week hypertensive grup were greater than cntrl (p < 0.05). The relatinship between PRA and the plasma aldsterne level f each grup was examined by linear regressin analysis. Whereas n significant crrelatin existed between these functins at 8 a.m., 4 p.m., r 12 MN in the cntrl grup, a significant crrelatin was present at each f these times within the fur hypertensive grups f rats (p < 0.01). A plt f PRA against plasma aldsterne cncentratin fr all rats in the hypertensive study revealed a high significant crrelatin (p < 0.001). The mean plasma sdium cncentratin was.5 ± 0.4 meq/liter and the mean plasma ptassium cncentratin was 4.05 ± 0.10 meq/liter fr the cntrl grup in bld cllected by cardiac puncture (table 1). Plasma sdium and ptassium cncentratins frm separate 8 a.m., 4 p.m. and 12 MN grups f nrmal as well as 4-, 5-, 7- and 10-week hypertensive rats did nt vary significantly amng the grups. Hwever, bld cllected by decapitatin frm the same cntrl rats cntained significantly higher plasma ptassium levels (4.77 ± 0.09 meq/ liter at 0-5 secnds after decapitatin and 5.40 ± 0.19 meq/liter at 5-15 secnds after decapitatin; p < 0.05 fr bth values cmpared with 4.05 ± 0.10 meq/liter fr the cntrl values). T evaluate mre fully the rle f the reninangitensin-aldsterne system in tw-kidney, ne TABLE 1. Plasma Sdium and Ptassium Cncentratins (meq/liter) in Nrmal Hypertensive and Rats Grup* Nrmals (n 6) Cardiac puncture Decapitatin 0-5 sees 5-15 sees 4-Week hypertensives (n = 9) 5-Week hypertensives (n - 9) 7-Week hypertensives (n = 9) 10-Week hypertensives (n = 7) Systlic arterial pressure (mm Hg) 108 ± ± ± ± ± 8 PN a.m. PK 4.04 ± ± ± ± ± ± ±0.15 PN. ±0.5 ±0.3 ± pi.m. PK 4.10 ± ± ± ± ± ± ±0.14 PN. 139 ± ± ± MN PK 3.85 ± ± ± ± ± ± ±0.10 *In the nrmal rats, bld was btained by cardiac puncture fr electrlyte analyses and then the animals were decapitated t btain additinal bld fr analyses. In the hypertensive rats, nly bld btained by cardiac puncture was analyzed.

6 CIRC ADI AN CHANGES IN HYPERTENSION/CrFmwr et al. 147 clip hypertensin in the rat, all rats sacrificed at 4 p.m. were divided int tw grups, a mderately hypertensive grup (arterial pressure < 180 mm Hg) and a severely hypertensive gtup (arterial pressure > 180 mm Hg). As presented in table 2, PRA f the mderately hypertensive grup was significantly elevated abve cntrl during Weeks 4 and 7, while the grup with severe hypertensin had a significantly higher PRA thrughut the study. In the grup with mderate hypertensin, plasma aldsterne cncentratin was elevated during Weeks 4, 5 and 10, while in the grup with severe hypertensin plasma aldsterne was elevated in all fur hypertensive grups. Discussin The present study is cncerned with the circadian changes in PRA and plasma aldsterne cncentratin in tw-kidney, ne clip hypertensin in the rat. There has been cnsiderable difficulty in assessing the functin f the renin-angitensin-aldsterne system in the rat and many cnflicting reprts have appeared. It is clear that anesthesia increases PRA" and it is almst impssible t btain reprducible basal plasma levels f renin and aldsterne cnsistently even in cnscius animals with chrnic, indwelling intravascular catheters. This prblem is evident frm the extensive studies in the spntaneusly hypertensive rat in which there is n unanimity f results n the plasma levels f renin and aldsterne; lw, 7 nrmal,* and high values' have been reprted. Ideally, an TABLE 2. Systlic Pressure, PRA, and PAC Measured at 4 p.m. in Nrmal Rats and in Rats with Mderate and Severe Hypertensin (HT) Grup Arterial pressure PRA PAC N. (mm Hg) (ng ang/ml/hr) (ng%) Nrmals ± ± ± 1.8 Hypertensives with systlic pressure belw 180 mm Hg 4 wks HT ± ± 0.8* 68 ± 38f 5 wks HT * ± * 4f 7 wks HT ± * 6.2f 22 ± 7 10 wks HT ± * ± 53f Hypertensives with systlic pressure abve 180 mm Hg 4 wks HT ± ± l.lf 57 ± 42f 5 wks HT ±8 4.3 ± 1.3f 69 *= 21f 7 wks HT ± ± 1.9f 102 ± 40f 10 wks HT ± ± 2.0f 111 ± 45t *p < tp < Abbreviatins: HT» hypertensin; PRA plasma renin activity; PAC = plasma aldsterne cncentratin; ang = angitensin I. average 24-hur value fr PRA and plasma aldsterne cncentratin is needed t assess what the effectr rgans respnd t daily. Als, circadian changes in renin and aldsterne have been reprted fr nrmal rats. 1 ' * These cnsideratins emphasize the serius limitatin f ne islated daily measurement f renin r aldsterne, r f the use f angitensin blckade at nly ne pint in time during a 24-hur perid t evaluate the rle f the reninangitensin-aldsterne system in the rat. Als, it is entirely pssible that varius experimental interventins r diseases alter the circadian peridicity f renin and aldsterne. The present results shw that bth PRA and plasma aldsterne cncentratin were higher at 4 p.m. than 8 a.m. r 12 MN in nrmal rats. T exclude an effect f anesthesia r ACTH, the animals were decpaitated t btain bld. There was n suggestin f seasnal variatins since the results btained in June and January were essentially the same. The present bservatins f peak values fr renin and aldsterne at 4 p.m. agree with earlierfindingsf Leenen et al. 1 and f Gmez-Sanchez et al. 1 Bth grups f wrkers demnstrated the highest PRA and plasma aldsterne levels during the afternn hurs. One factr cntributing t the circadian variatins in the rat is the ncturnal eating habit with increased sdium intake which wuld lead t lw renin and aldsterne values fr the samples taken at 12 MN and 8 a.m. These findings in the rat are in cntrast t results btained in humans in whm the highest values fr bth PRA and plasma aldsterne cncentratin ccurred between 12 MN and 8 a.m. 10 ' n These changes appeared t be independent f any f the knwn mechanisms fr cntrl f renin and aldsterne secretin." " With assumptin f the upright psture and stimulatin f the renal sympathetic nerves, PRA increased between 8 a.m. and 12 nn."' 1S The present study is the first investigatin f circadian changes in PRA and plasma aldsterne cncentratin in experimental renal hypertensin. Althugh PRA was elevated in hypertensive animals frm 4 t 10 weeks, the circadian variatin present in nrmals with a peak value at 4 p.m. had disappeared. It is ntewrthy that the circadian peridicity in PRA als disappeared during sdium depletin in the rat. 2 The excessive stimulatin f imprtant cntrl mechanisms fr renin release such as the renal vascular receptr and the macula densa might explain the lss f the circadian rhythm fr PRA in hypertensive animals. There is a paucity f data n aldsterne secretin r plasma aldsterne cncentratin in tw-kidney hypertensin in the rat. In 1960, MOller and Grss 14 reprted that adrenal tissue frm 4-week tw-kidney hypertensive rats prduced mre aldsterne in vitr than adrenal tissue frm nrmal animals. In 1963, Singer and assciates 16 fund that aldsterne secretin was increased in anesthetized tw-kidney rats with hypertensin f weeks duratin; crticsterne was nt detectably changed. The present

7 148 HYPERTENSION VOL 1, N 2, MARCH-APRIL 1979 bservatins demnstrate that the plasma aldsterne cncentratin was elevated after 4-, 5-, 7- and 10 weeks f hypertensin. Furthermre, the circadian peridicity fr aldsterne was nt nly sustained in the tw-kidney hypertensive rats, but the peak value at 4 p.m. appeared t be mre exaggerated in the 4-, 5-, and 10-week grups than in nrmal animals. The present findings suggest that factrs ther than PRA might have cntributed t the elevated 4 p.m. values fr plasma aldsterne cncentratin since the circadian rhythm fr PRA was lst in the hypertensive animals. Plasma electrlyte cncentratins were studied in an attempt t explain the circadian changes in aldsterne. When bld was cllected by decapitatin, the values fr plasma ptassium cncentratin were strikingly elevated; apparently, hwever, this elevatin resulted frm changes assciated with the prcedure fr cllectin f bld. When bld was cllected by cardiac puncture, bth plasma ptassium and sdium cncentratins shwed n evidence f circadian changes and were nt altered in hypertensive animals. It is pssible that changes in plasma ACTH cntributed t the circadian rhythm in plasma aldsterne but n attempt was made t evaluate a rle fr ACTH. Althugh factrs ther than PRA culd be invlved in the cntrl f plasma aldsterne cncentratin, it is clear frm the present high crrelatin cefficient between the plasma levels fr renin and aldsterne that PRA is a very imprtant determinant f the plasma aldsterne cncentratin in the twkidney hypertensive rat. There is cnsiderable cnfusin in the literature n the rle f the renin-angitensin system in the pathgenesis f tw-kidney hypertensin in the rat. Evidence gathered frm studies with either angitensin II blcking agents'-" * r actual measurements f PRA 3 ' " M have indicated that the renin-angitensin system is hyperactive during the acute phase (1-6 weeks) f hypertensin' 1 lm0 but nrmal during the chrnic phase (15-16 weeks) f the disease.'- 18 Hwever, in tw studies in anesthetized rats, 1 '- 21 it has been reprted that drps in bld pressure ccurred with angitensin II analgues after 4 mnths f hypertensin. The present data n the circadian peridicity f bth renin and aldsterne indicate that an evaluatin f the renin-angitensin-aldsterne system might be mre cmplete if measurements were made at least three times during the day. Als, the present results pint t the desirability f making studies in cntrl and experimental animals at the same time f day. Failure f varius wrkers t d this, alng with the use f anesthesia which increases PRA in the rat, may accunt fr much f the variatin in reprted results. In the present study, PRA had nt returned t nrmal by 10 weeks after clipping the renal artery. Carreter and Gulati' have recently suggested that activatin f the renin-angitensin system in tw-kidney hypertensive rats is nt nly dependent n the time perid fllwing renal artery cnstrictin, but als n the severity f the hypertensin. It has previusly been demnstrated that placing a 0.2-mm clip n the renal artery will prduce bth benign and s-called malignant hypertensin in tw-kidney rats."- m Whereas the benign hypertensive animals had a high PRA nly during the initial phase f hypertensin,' the rats with severe hypertensin had an elevated PRA in assciatin with a negative sdium balance and a very high bld pressure during the chrnic stage f the disease."- " The present findings revealed that rats with mderate hypertensin f 5- and 10-weeks duratin had a nrmal PRA whereas at 4 and 7 weeks PRA was elevated; the rats with severe hypertensin had an elevated PRA thrughut the study. Only five rats in the grup with severe hypertensin were in a negative sdium balance. These cnsideratins suggest, therefre, that the rle f the renin-angitensin system in the pathgenesis f tw-kidney hypertensin in the rat is related t bth the duratin and the severity f the hypertensin. References 1. Leencn FH, Scherren JW, Omylanwski D, Elema JD, Vanderwal B, De Jng W: Changes in the renin-angitensinaldsterne system and in sdium and ptassium balance during develpment f renal hypertensin in rats. Clin Sci Ml Med 48: 17, Gmez-Sanchez C, Hlland OB, Higgins JR, Kem DC, Kaplan NM: Circadian rhythms f serum renin activity and serum crticsterne, prlactin, and aldsterne cncentratin in the male rat n nrmal and lw-sdium diets. Endcrinlgy 99: 567, Carreter OA, Gulati OP: Effects f angitensin antagnist in rats with acute, subacutc and chrnic tw-kidney renal hypertensin. J Lab Clin Med 91: 264, Sealey JE, Laragh JH, Gcrten-Bane J, Acct RM: The measurement f plasma renin activity in man. In Hypertensin Manual, Yrke Medical Bks, New Yrk, Dun-Dnnelley Publishing Crp, 1974, p Buhlcr FR, Sealey JE, Laragh JH: Radiimmunassay f plasma aldsterne. In Hypertensin Manual, Yrke Medical Bks, New Yrk, Dun-Dnnelley Publishing Crp, 1974, p Pettinger WA, Tanaka K, Keetn K, Campbell WB, Brks SN: Renin release, an artifact f anesthesia and its implicatins in the rat. Prc Sc Exp Bil Med 143: 625, Freeman RH, Davis JO, Varsan-Anarn N, Ulick S, Weinberger MG: Cntrl f aldstcrne secretin in the spntaneusly hypertensive rat. Circ Res 37: 66, Frman BH, Mulrw PJ: Effect f prpranll n bld pressure and plasma renin activity in the spntaneusly hypertensive rat. Circ Res 35: 215, Sen S, Smeby RR, Bumpus FM: Renin in rats with spntaneus hypertensin. Circ Res 31: 876, Grdn RD, Wlfe LK, Island DP, Liddle GW: A diurnal rhythm in plasma renin activity in man. J Clin Invest 45: 1587, Mdlingcr RS, Sharif-Zedeh K, Ertel NG, Gutkin M: The circadian rhythm f renin. 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8 ORCADIAN CHANGES IN HYPERTENSION/fleFrrw/ et al Mflller J, Grss F: Effects f experimental renal hypertensin n aldsterne bisynthesis by rat adrenal tissue. Acta Endcrinl 60: 669, Singer B, Lsit C, Salmn S: Aldsterne and crticsterne secretin rates in rats with experimental renal hypertensin. Acta Endcrinl 44: 505, Pals DT, Masucci FD, Denning GS, Sips F, Fessler DC: Rle f the pressr actin f angitensin II in experimental hypertensin. Circ Res 29: 673, Bumpus FM, Sen S, Smeby RR, Sweet C, Ferrari CM, Khsla MC: Use f angitensin II antagnists in experimental hypertensin. Circ Res 32, 33 (suppl I): 1-150, Gavras H, Brunner HR, Thurstn H, Laragh JH: Reciprcatin f renin dependency with sdium vlume dependency in renal hypertensin. Science 188: 1316, Thurstn H, Swales JD: The rle f sdium retentin in Gldblatt tw-kidney hypertensin in the rat. Clin Sci Ml Med 51: 1255, Hutchinsn JS, Matthews PG, Dax E, Jhnstn CF: Plasma renin and angitensin levels in experimental hypertensin in the rat. Clin Exp Pharm Physil Suppl 2: 83, Bing J, Nielsn K: Rle f the renin system in nrm- and hypertensin. Acta Path Micrbil Scand [A] 81: 254, Mhring J, MShring B, Petri M, Haack D, Hackenthal E: Studies n the pathgenesis f the malignant cause f renal hypertensin in rats. Kidney Int 8: S174, MChring J, MShring B, Naumann HJ, Philippi A, Hmsy G, Orth H, Danda G, Kazda S, Grss F: Salt and water balance and renin activity in renal hypertensin f rats. Am J Physil 228: 1847, 1975