STUDIES ON THE MECHANISM OF RENOVASCULAR HYPERTENSI'ON Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY RAUL CARDONA, M. D. 1.976 " LIBRARY ' Eficiflgm State University ABSTRACT STUDIES ON THE MECHANISM OF RENOVASCULAR HYPERTENSION BY Raul Cardona, M.D. The etiology of renovascular hypertension (RVHPT) has been studied for many years. After the renin-angiotensin system was discovered, several studies showed that this system may be the main cause of this type of hypertension. However, in the last decade, several investigators have reported some evidences which support the hypothesis that another pressor substance or system may play role in the etiology of RVHPT. The objectives of this thesis work were: (1) To test the hypothesis that during the chronic stage of RVHPT unknown humoral factor(s), different from, but not necessarily independent of the renin-angiotensin-aldosterone system, is (are) involved; and (2) To determine certain physicochemical and/or biological characteristics of that factor. Three groups of experiments were attempted. First, studies were performed to find out if chronic (four or more Raul Cardona weeks after hypertension) one-kidney Goldblatt type hypertension in rats (l-KHR) is related to plasma renin activity (PRA) levels. There was no significant correlation between blood pressure (BP) and PRA (correlation coefficient: r = 0.002, n = 27, p > 0.05). It was also found that in one-kidney perinephritis hypertension in dogs (l-KHD) there was a significant suppression of both PRA and aldosterone concentration (p < 0.05). After three weeks of hypertension, aldosterone concentration became normal but PRA was still low and it remained so for three months after the development of hypertension. ‘ Secondly, it was attempted to determine the effect of the plasma obtained from normotensive and hypertensive rats and dogs, on the pressor activity of angiotensin II (A11) and norepinephrine (NE). Blood pressure (BP) response of bilaterally nephrectomized-pentolinium treated rats (Bioassay I) was used as model to study the effect of the plasma on AII vasopressor activity. There was an increased response to AII after injection of plasma from hypertensive, but not after the injection of plasma from normotensive rats. The maximum effect was usually seen between 20 to 40 minutes after plasma administration. Additionally, isolated mesentery arteriole (Bioassay II) was used to study the effect of the plasma, obtained from hypertensive dogs, on the vasopressor activity of NE. The dose-response curve of Raul Cardona NE was significantly increased (p < 0.05) during perfusion with the plasma from hypertensive dogs. Thirdly, two types of fractionation were done to determine whether any specific fraction may increase the vasopressor activity of AII in Bioassay I. Plasma was separated into four main fractions by using Amicon Diaflo Ultramembranes: (l) UM-10 retainer, substances with molecular weight more than 10,000. This fraction was not tested because of the very high concentration of large molecules; (2) UM-Z retainer, substances with molecular weight between 1,000 and 10,000; (3) UM-OS retainer, substances with molecular weight between 500 and 1,000; and (4) UM-05 filtrate, substances with molecular weight less than 500. In initial experiments, fraction 4 failed to affect the pressor activity of A11 in Bioassay I and, for this reason, this fraction was not used in any further study. Fractions 2 and 3 enhanced the action of A11 in Bioassay I; the effect of fraction 3 was significantly higher than that observed with fraction 2 (p < 0.05). UM-05 retainer was further fractionated on Bio-Gel P—2 column (1.5 x 90 cm) and 6 m1 eluate was collected in each tube. Fraction No. 20 (115 to 120 m1 of eluted volume) from l—KHD produced a significant increase of A11 vasopressor activity, but no effect was observed with fraction.No. 20 from normotensive dogs. These tractions contain substances with molecular weight close to 1,000. Raul Cardona It is concluded that a humoral factor of small molecular weight may exist during one-kidney renovascular hypertension and it may play an important role in maintaining the high blood pressure by increasing the pressor activity of angiotensin II and norepinephrine. STUDIES ON THE MECHANISM OF RENOVASCULAR HYPERTENSION by Raul Cardona, M.D. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacoloqy 1976 ACKNOWLEDGEMENTS I would like to thank Dr. Andrew M. Michelakis, my advisor, for his guidance and encouragement during my graduate study work. I am indebted to Dr. Theodore M. Brody, the Chairman of the Department of Pharmacology, for his interest in my research. I appreciate the generous constructive criticism of my graduate committee members: Dr. Tai Akera and Dr. Ching-Chung Chou. I would like to acknowledge the help from all the postdoctoral personnel at the Clinical Pharmacology Unit of the Department of Pharmacology. I am specially grateful to Dr. Chan-Ting Huang for his invaluable and worthwhile suggestions. The technical assistance of Mr. Paul Susan and Ms. Ivy Mao is highly appreciated. ii ACKNOWLEDGEMENTS. . TABLE OF CONTENTS . LIST OF FIGURES . . INTRODUCTION. . . . l. 2. General background . Main factors related TABLE OF CONTENTS to increas resistance Renovascular Hypertension . . SPECIFIC OBJECTIVES . . . . . . MATERIALS AND METHODS . . . . . 1. 2. 3. 7. 8. RESULTS 1. Clamping the renal artery in rats. wrapping the kidney in dogs. . . and blood Blood pressure measurement collection . . . . . . a. Rats . b. Dogs . Plasma fractionation b. Gel filtration . a. Ultrafiltration. . Radioimmunoassays. . a. Plasma renin activity. b. Aldosterone. . . . Bioassays. a. Blood pressure assay in rats b. Isolated mesenteric arteriole. Definitions. . . . . . Statistical analysis . Relationship among plasma renin activity and aldosterone concentration and blood pressure in one-kidney Renovascular Hypertension . . . . . iii ed vascular 23 TABLE OF CONTENTS CONTINUED Effect of plasma on the activity of angiotensin II on blood pressure of ganglion-blocked and bilaterally nephrectomized rats. . . . . . . . . . . . . . Effect of plasma on the pressor response of the isolated mesenteric arteriole to norepinephrine . . . . . . . . . . . . . . . . Effect of plasma fractions on the activity of angiotensin II on blood pressure of ganglion-blocked and bilaterally nephrectomized rats. . . . . . . . . . . . . . DISCUSSION. 0 O O O O O O O O O O O O O O O O O O O O O l. 2. Relationship between renin-angiotensin- aldosterone system and one-kidney Renovascular Hypertension. . . . . . Evidence for the presence of a factor which potentiates the action of angiotensin II and norepinephrine in one-kidney Renovascular Hypertension . . . . . . . . . . . . . a. Effect of plasma from normotensive and hypertensive rats and dogs on vascular response to angiotensin II and norepinephrine . . . . . . . . . . . . b. Effect of plasma fractions from normotensive and hypertensive dogs on vascular response to angiotensin II . . . . . . . . . . . . . . s UMMRY AND CONCLUS ION S O O C O O O U 0 O O 0 O O O O O BIBLIOGMPHY. O O O O O O O O O O O O O O O O O O O O 0 iv 35 43 48 59 59 61 61 63 65 66 Figure LIST OF FIGURES Title Page Effect of wrapping the left kidney in uninephrectomized dogs on blood pressure, plasma renin activity and aldosterone concentration. . . . . . . . . . . . . . . . . 28 Relationship between blood pressure and plasma renin activity in dogs during the chronic stage of one-kidney perinephritis hypertension . . . . . . . . . . 30 A. Blood pressure in groups of randomly chosen one-kidney hypertensive and sham operated rats over a nine-week period . . .'. . . . . . . . . . . . . . . 32 B. Plasma renin activity in groups of randomly chosen one-kidney hypertensive and sham operated rats over a nine-week period. . . . . . . . . . 32 C. Blood pressure in a group of four one-kidney hypertensive rats over a nine-week period . . . . . . . . . . . . 32 Relationship between blood pressure and plasma renin activity in chronic one-kidney hypertensive rats . . . . . . . . . 34 Typical response of the blood pressure of bilaterally nephrectomized rats to the intravenous injection of angiotensin II before and after the administration of plasma from chronic one-kidney hypertensive and normotensive rats. . . . . . . . . . . . . 33 Effect of plasma from chronic one-kidney hypertensive and normotensive rats on the vasopressor activity of angiotensin II in ganglion-blocked and bilaterally nephrectomized rat . . . . . . . . . . . . . . 4° Figure 10 ll 12 13 LIST OF FIGURES CONTINUED Title The dose-response curve for plasma from chronic one-kidney hypertensive and normotensive rats on the pressor response of ganglion-blocked and nephrectomized rat to angiotensin II. . . . . . . . . . . . Typical response of the perfusion pressure of isolated mesenteric arteriole to different doses of norepinephrine during the perfusion of Krebs buffer solution, and plasma from chronic one-kidney perinephritis hypertensive and normotensive dogs in buffer solution . . . . The dose-response curve for NE on the isolated mesenteric arteriole during the perfusion with plasma from chronic one- kidney perinephritis hypertensive and normotensive dogs in Krebs buffer solution . Effect of plasma fractions (ultramembrane filtration) from chronic one-kidney perinephritis hypertensive dOgs on the vasopressor activity of All in ganglion- blocked and bilaterally nephrectomized rat . Effect of plasma fractions (gel filtration) from chronic one-kidney perinephritis dogs on the vasopressor activity of All in ganglion-blocked and bilaterally nephrectomized rat . . . . . . . . . . . . . Effect of plasma fractions (gel filtration) from normotensive dogs on the vasopressor activity of A11 in ganglion-blocked and bilaterally nephrectomized rat . . . . . . . Effect of plasma fraction No. 20 (Bio-Gel P-2 column) from chronic one-kidney perinephritis hypertensive and normotensive dogs on the vasopressor activity of A11 in ganglion-blocked and bilaterally nephrecto- mized rat. . . . . . . . . . . . . . . . . . vi Page 42 45 47 52 54 56 58 INTRODUCTION 1. General background After Goldblatt's studies (1934, 1937) on experimental reno-vascular hypertension, and the description of angiotensin by Page gt_gl., (1940) and Braun-Menendez gt_gl., (1940), the study on the pathogenesis of RVHPT has been centered on the renin-angiotensin-aldosterone system (R-A system). The following is a simplified scheme on the main points of this system: Angiotensinogen (a2 globulin) renin Angiotensin I (decapeptide) converting enzyme Angiotensin II (octapeptide) ? peptidases Angiotensin III (heptapeptide) By-products Angiotensin II (AII), on one hand produces a direct smooth muscle contraction, probably by acting on Ca++ firmly bound to the membrane and increasing the free Ca++ inside the cell (Somlyo and Somlyo, 1970). On the other hand, AII increases the release of aldosterone (Gross, 1968), and catecholamines (Douglas §t_al,, 1967) from adrenal glands. In addition, it has been reported that AII may act on the central sympathetic system (Severs and Daniels-Severs, 1973) and also on sympathetic nerve terminals (Distler §E_al., 1965). Peripherally, this peptide may increase the catecholamine levels either by releasing norepinephrine from nerve endings (Gascon and Walaszek, 1968), by blocking the reuptake of this neurotransmitter (Khairallah gE_gl., 1971) or by accelerating the synthesis of norepinephrine from precursors (Davila 33:11., 1971). It appears that the R-A system is essential in the circulatory homeostasis in normotensive animals. Although the R-A system seems to play an undoubtedly very important role in RVHPT, some experimental data have raised the question of whether or not it is the only contributor in this type of hypertension. Shortly after the Goldbatt studies, it was reported that in dogs with RVHPT in the chronic state, the blood pressure (BP) may remain high even though the plasma renin levels are usually normal (Haynes and Dexter, 1947). This finding has been confirmed by others (Blair-West et al., 1968; Harris and i[[‘ | ‘lllll‘ l 1'. l- Ayers, 1972). The same non-relationship between experimental chronic RVHPT and angiotensin levels has been reported by several workers (Brunner EE_2l" 1972; Scormik and Paladin, 1961). Furthermore some investigators have not found a consistent relationship between PRA and BP in clinical RVHPT (Bath gg_gl., 1968). Also, aldosterone secretion is usually normal in patients with unilateral renal hypertension (Laragh gg_31., 1966). It is generally accepted that RVHPT can be divided into acute and chronic phases. These two phases may exhibit differences in their relationship with the R-A system depending upon the experimental design. In one-kidney renal hypertension (l-KH), which can be done by clamping one renal artery and contralateral nephrectomy (Goldblatt gt_al., 1934) or by wrapping one kidney and nephrectomy (Page, 1939), PRA have been found to be either normal (Helmchen et al., 1972b), low (Mogil gt_§1., 1969), or high (Ayers gt_§1., 1969) in the acute phase. However, after two or three weeks (chronic phase) PRA levels are often normal (Brown g£_§1,, 1966; Koletsky and Rivera-Velez, 1967; Fujii and Ikeda, 1971; Liards and Peters, 1973). Furthermore, AII inhibitors (Bumpus gt_31., 1973; Skeggs gt_gl., 1975), converting enzyme inhibitors (Engel g£_31,, 1973), and immunization against AII (Eide and Aars, 1969; Johnston and Mendelson, 1970; Louis gt_31., 1970), in many instances could not influence the high BP in chronic l-KH. Also, active immunization with "renin" (renal extracts) have decreased the BP in some chronic l-KH (Wakerlin §£_21,, 1958) but not in others (Hill gt_§1., 1970). The fact that impure renin extracts have been used in those studies makes it difficult to interpret these findings. Skeggs §E_gl., (1976) have been able to obtain a kidney cortex preparation with little or no renin; active immunization with that extract lowered the BP in rabbits with chronic l-KH, although there was no formation of plasma anti-renin. These authors produced high plasma anti-renin titers in another group of 1-K hypertensive rabbits without affecting the BP levels. In two-kidney renal hypertension (2-KH), which can be done by clamping one renal artery or wrapping one kidney and leaving the contralateral organ intact (Skulan et al., 1974; Thurston and Swales, 1974; Liard and Peters, 1973), or by clamping both renal arteries (Wakerlin et al., 1958), PRA is usually increased in both acute and chronic phase (Skulan et al., 1974). Moreover, AII inhibitors (Bumpus gt_§1., 1973) or anti-angiotensin antibodies (Brunner g£_al., 1971) can decrease the BP in chronic 2-K RVHPT. Several reviews have supported a significant relationship between PRA and BP in 2-K hypertension (Liard and Peters, 1973; Oparil and Heber, 1974; Laragh et al., 1975). 2. Main factors related to increased vascular resistance in Renovascular Hypertension A. Fixed Factors 1) Increased in the wall to lumen ratio (Folkow gt 31., 1958; Folkow and Neil, 1971; Sivertson and Olander, 1968). The initial increase in BP, which may be due to a high PRA or an early increase in cardiac output (Ferrario, 1974), causes generalized hypertrophy of the vascular wall of the arterioles and therefore, produces an increase in the wall to lumen ratio. As it has been proposed (Folkow g5_gl., 1958), the higher the wall to lumen ratio, the higher the reduction in luminal diameter (or the higher the vascular resistance) for any degree of smooth muscle shortening. Even though this mechanism may play a significant role, it fails to explain, for example, the fall in blood pressure after removing the renal artery constriction in animals with low renin chronic RVHPT as it has been reported in sheep (Blair-West gt_gl., 1970), dogs (Tagawa gt_§1., 1974) and rats (Neubig and Hoobler, 1975). 2) Water and electrolytes retention: Some investigators have found an increased amount of water and electrolytes (Na+) in the wall of both large (aorta) and small (mesenteric arterioles) vessels from hypertensive animals and/or humans (Tobian and Binion, 1952, 1954; Tobian et al., 1961; Tabian et al., 1969). On one hand, the electrolyte changes could alter the vascular response by acting at some levels of the excitation-contraction coupling of vascular smooth muscle (Jones, 1974), and on the other, the water effect would be indirect, mainly by altering the wall to lumen ratio (Tobian et al., 1969). 3) Enhanced vascular response due to an increase in adrenergic nerve innervation: It has been reported that during hypertension, the adrenergic innervation of blood vessels as well as synthesis of NE could be enhanced (Bevan gt_al., 1974; Bevan gt_al., 1975; De Quattro and Alexander, 1974; Samir Amev gt_31., 1975). However, it is not clear what the actual reson for these changes would be. B. Humoral Factors During the last 10 years, some investigators have raised the possibility that an unknown renal substance may be responsible for the maintenance of chronic RVHPT (Genest §E_31., 1964; Skeggs gt_gl., 1975, 1976). Recently, it has been suggested that one pressor substance different from renin and angiotensin is present in the acute phase of RVHPT (Grollman, 1970; Grollman and Krishnamurty, 1971; Susic and Sparks, 1975); it was called nephrotensin. However, studies brought about by others have related nephrotensin to angiotensin I (Schweikert gg_§l., 1972). Mizukoshi and Michelakis (1972) have found that peripheral venous plasma from chronic hypertensive patients (PVP) has a moderate pressor effect and also potentiates the activity of AII and NE on the blood pressure of small (body weight 180 to 200 gm), ganglion-blocked and bilaterally nephrectomized rats. Greenberg §t_gl. (1974) reported that in big rats (body weight 525 to 585), neither pretreated with pentolinium nor nephrectomized, PVP enhanced the pressor response to tyramine, but did not affect the pressor response to AII and NE. The fact that the Greenberg's study was done with old rats may be important in the difference observed with Mizukoshi and Michelakis' report as it has been shown that receptors to catecholamines decrease with increasing age (Fleish gt_§l., 1970; Fleish, 1971). Also, in Greenberg's study animals were neither pretreated with pentolinium nor nephrectomized; therefore, rats could have been able to compensate BP changes after plasma administration and to excrete rapidly the injected substance by the urine. Recently, it has been reported that plasma fractions from one-kidney hypertensive dogs can potentiate the pressor activity of AII and NE (Michelakis et al., 1975). SPECIFIC OBJECTIVES The objectives underlying this thesis work were: (1) To determine if the chronic one-kidney hypertension is related to plasma renin activity and aldosterone concentration; (2) To determine whether an unknown circulating factor is presented in chronic one-kidney hypertension; and (3) To attempt to determine approximated molecular weight, stability and pharmacodynamics of that factor. MATERIALS AND METHOD S 1. Clamping the renal artery in rats Sprague Dawley male rats weighing between 180 to 200 gm were used. Under sodium pentobarbital anesthesia (35 mg/kg., i.p.), a dorsal incision of about 2 cm was made to expose the left kidney and the ipsilateral renal artery was partially isolated. For clamping the artery, the Schaffenburg's method (1959) was used. Clips (bent strips of fine silver, 0.005 inches thick, 6 x 2 mm) were applied with a calibrated forceps (0.2 mm i.d.). Another incision was made on the other side and the right kidney was removed. Incisions were closed with separate suturing of muscle and skin. In the sham Operated rats, one kidney was removed but the renal artery of the other kidney was left without clamping. All animals in both groups had free access to commercial rat food (wane-Lab-Blox) and tap water. 2. Wrapping the kidney in dogs Male, young and healthy dogs weighing about 18 kg were anesthetized with sodium pentobarbital (25 mg/kg., i.v.) and under sterile conditions a flank incision of about 12 cm was 9 10 made and the left kidney was wrapped in a silk bag. After one week, another incision was performed on the other side and the right kidney was removed. After surgery, dogs received penicillin (100,000 units) and streptomycin (0.1 gm) i.m. daily for 5 days. They had a free access to commercial dog food and tap water. 3. Blood pressure measurement and blood collection a. Rats: Systolic BP was measured in conscious rats. They were warmed at 45°C in a box for a 5 minute period. Afterward, rats were removed from the box and placed in a restraining cage (Narco-Bio Systems, Inc.), where a cuff was put on the proximal part of the tail and the BP was measured under heating (40°C) and after allowing 4 to 5 minutes for stabilization. Pneumatic pulse transducer, Electrosphygmograph ESG 3000 and Physiograph Desk Model DMP 4A (Narco-Bio Systems, Inc.) were used in BP measurement. Blood collection in rats was done under sodium pentobarbital anesthesia (35 mg/kg., i.p.). About 10 m1 of arterial blood samples were withdrawn from the carotid artery in anesthetized rats, and the blood sample was placed in a cold plastic centrifuge tube which contained 5 mg of disodium-EDTA (disodium ethylenediamine tetracetate), and was centrifuged for 20 minutes at 3,000 r.p.m. Plasma was kept at -20°C until assay. 11 b. Dogs: Male, young and healthy dogs weighing about 18 kg were trained for at least four weeks in order to take BP directly from a femoral artery in conscious and calmed dogs, using a transducer P23DC Statham and Grass Model 70 Polygraph. BP was measured once every week before and after surgery. After blood pressure determination, about 20 m1 of venous plasma was withdrawn from the jugular vein, blood was placed in a cold siliconized glass tube which contained 101mg of disodium-EDTA, and centrifuged for 20 minutes at 3,000 r.p.m. Plasma was kept at -20°C until assay. 4. Plasma fractionation a. Ultrafiltration: Under an applied pressure (30 1b/ sq. in.) of N2 atmosphere, 50 m1 of plasma was filtrated through Amicon Diaflo Ultramembrane 10 (UM-10). The filtrated solution was passed through UM-2, and the filtrated solution from UM-2 was further filtrated on UM-05. The retainers from UM-2 and UM-05 were dissolved in 5 m1 of 0.9% NaCl and kept under N2 atmosphere at -20°C until assay (not more than 3 days). At the end, four fractions were obtained: UM-10 retainer Molecular weight > 10,000 UM-2 retainer Molecular weight > 1,000, <10,000 UM—05 retainer Molecular weight > 500, ’0.05). b. Rats: Sixty Sprague Dawley rats weighing between 180 to 200 gm were used. Under sodium pentobarbital anesthesia (35 mg/Kg., i.p.), the left renal artery was clamped and, in the same operation time, the right kidney was removed. BP was significantly higher (p < 0.05) than the pre-operative values one week after the surgery (Fig. 3A. Close circles). After the second week of hypertension, we randomly chose a group of hypertensive rats every week; the BP values of those groups (Fig. 3A. Close circles) were significantly higher (p < 0.05) than those of sham operated rats (Fig. 3A. Open circles) during the entire period of observation. The group of randomly chosen hypertensive rats, and also a group of sham operated rats, were sacrified every week, 24 hours after the BP determination, to obtain the plasma. PRA showed no significant changes during the nine week period either in hypertensive (Fig. 3B. Close circles) or normotensive (Fig. BB. Open circles) rats. Additionally, a group of four hypertensive rats was followed over a nine week period to figure out the usual development of BP in one-kidney hypertensive rats. The control BP of this group was 12535.0 mm/Hg (SEM); two weeks after the operation the BP value was significantly higher than those before surgery (p < 0.05) and then BP constantly increased up to the end of the period of study (Fig. 3C). 26 In order to evaluate the relationship between BP and PRA in the chronic stage of l-KHR, correlation analysis was performed by the least squares method (Fig. 4). There was no significant correlation between BP and PRA (r = 0.002, n = 27, p > 0.05). Clamping one renal artery in contralaterally nephrectomized rats had a significant effect on BP level but did not effect PRA, suggesting that renin-angiotensin system does not play a significant role in the maintenance of BP in chronic one-kidney hypertension. Figure l. 27 Effect of wrapping the left kidney in uninephrectomized dOgS on: Blood pressure (BP, n = 5); Plasma renin activity (PRA, n = 4), and Aldosterone concentration (Aldo, n = 3). Vertical lines represent standard error of the mean (SEM). (*) Significantly different compared to pre-operative values (p < 0.05). 28 l.;\_s\uev .52 I2 I 1 ll Wee ks m .uOLOQO 1.0.. Eco I Figure Figure 2. 29 Relationship between BP and PRA in four dogs during the chronic stage of one-kidney perinephritis hypertension. There was a significant negative correlation between BP and PRA (r = 0.785, n = 12, p < 0.05). Correlation coefficient was calculated by the least squares method. 30 PLASMA RENIN ACTIVITY (ng/ml/hr.) OI A 01 N O wa .wwwnom .oo .8 ~00 2:000 tmmmmcmm “3....(13 . IDES 9 Figure 3. A. 31 Mean value of blood pressure (SEM) in one- kidney hypertensive (close circles, n = 2 to 13) and sham operated (open circles, n = 2 to 10) rats over a nine-week period after operation. Points without SEM are the mean value of 2 rats. (*) Significantly different compared to preoperative values (p < 0.05). Mean value of plasma renin activity (SEM) in one-kidney hypertensive (close circles, n = 2 to 13) and sham operated (open circles, n = 1 or 2) rats over a nine-week period after operation. Points without SEM are the value of l or the mean value of 2 rats. There were no significant changes during the entire period of observation. Mean value of blood pressure (SEM) in a group of four hypertensive rats over a nine-week (period. (*) Significantly different compared to preoperative values (Op. means operation time). 25 P R A (mg/ml/hr) U! 9 240 B P (”T/H9) I20— Figure 3 Figure 4. 33 Relationship between BP and PRA in 27 rats during the chronic one-kidney hypertension. There was no significant correlation (r = 0.002, n = 27, p > 0.05). Correlation coefficient was calculated by the least squares method. Open circles represent values from sham operated rats. Close circles represent values from one-kidney hypertensive rats. -.—-—-=-—-. ‘—___ . 34 we (ng/ml/hr.) 6‘1 8 8 PLASMA RENIN ACTIVITY 8 Loo .. u obom oV Pom O C o a O O O O O O m o o U m o _ F n e P L 30 Moo Nmo erOU pmmmmcmm 3:118 39.3 b 35 2. Effect of plasma on the activity of angiotensin II on blood pressure of ganglion-blocked and bilaterally nephrectomized rats In the first serie of Bioassay I, we studied the change in the response of the assay rat to a constant amount of AII after the administration of plasma from normotensive and hypertensive rats. There was a rise in mean BP (base line) and vasopressor response to All after intravenous injection of 20 Ml or 50 ul of plasma from hypertensive but not from normotensive rats. Figure 5 shows a typical response of the assay. Vascular responsiveness to AII usually increased 10 to 15 minutes after the administration of plasma from l-KHR. The maximal increase was seen 20 to 25 minutes after the injection of plasma, and the effect continued for 30 to 60 minutes (Fig. 6). The difference between the values obtained with plasma from hypertensive and normotensive rats was statistically significant (p < 0.05) 10, 15, 20 and 25 minutes after the injection of plasma. In the second serie of experiments, we evaluated whether the effect of plasma on the pressor activity of AII in Bioassay I exhibits a dose-response relationship. A constant dose of AII was intravenously injected every 5 to 10 minutes after the administration (i.v.) of 5, 10, 15, 17, 20 and 50 ul of plasma per 130 gm rat. Ten microliters of plasma from l—KHR was the minimal amount which could enhance the vascular response to AII. Maximal plasma effect was 36 obtained with 20 pl or 50 ul of plasma from l-KHR. Plasma from normotensive rats did not produce any significant increase in the response of this bioassay to AII (Fig. 7). Figure 5. 37 Typical response of the blood pressure of bilaterally nephrectomized rats to the intravenous injection of angiotensin II (AII) before and after the administration of plasma from chronic one-kidney hypertensive (HPT) and normotensive rats (NT). There was a rise in both mean blood pressure (baseline) and response to angiotensin II after injection of plasma from hypertensive but not from normotensive rats. —.“_-... au- _ fl 38 Pressure (m m/Hg) Blood So I 8.. a a a \_, \_, \_, >__ It.— Bow—so .ooI SI 9 s ._, >__ ' 8&33 Zq Ecuao F _ _. _ _ _ b 1313:; o 5 no so .8 mo 00 Inc; u Figure 6. 39 Effect of plasma from chronic one-kidney hypertensive (0-0) and normotensive (o-o) rats on angiotensin II vasopressor activity in bilaterally nephrectomized rats. Each point is the mean of 13 values. Vertical lines represent the standard error of the mean. (*) Significantly higher (p < 0.05) than the effect obtained after the administration of plasma from normotensive rats. 40 PERCENT CHANGE IN AII RESPONSE ABOVE CONTROL mcj bol war. we .5 I In... 23.3 0'0 2... 233m m 8 a Mo Na 8 .2223558 2.84 Emmam magmamfimzoi Inc-d O 41 Figure 7. The dose-response relationship of the percent difference between the rise in BP of bilaterally nephrectomized rats caused by AII before and after the injection of different amounts of plasma from chronic one-kidney hypertensive (o-o) and normotensive (o-o) rats. Each point is the mean of three experiments. Vertical lines represent the standard error of the mean. (*) Significantly higher (p < 0.05) than the effect obtained after the administration of plasma from normotensive rats. 42 no I I It... 233» 0'0 2... 2338 w o I E 3 PERCENT CHANGE IN AII RESPONSE ABOVE CONTROL N o I _| O I o I I _ _ _ . do dmduNo mo t. O." vr>m§> 3926 N 01 43 3. Effect of plasma on the pressor response of the isolated mesenteric arteriole to norepinephrine The dose-response curve for NE during perfusion with Krebs buffer solution was studied. At basal perfusion pressure close to 30 mm/Hg the threshold dose of NE was often 50 ng in bolus injection. We could not determine the maximal response of the assay to NE because this catecholamine usually produced an out of scale increase of perfusion pressure with any dose above 300 ng per preparation. Figure 8 shows a typical response of Bioassay II to NE during the perfusion of Krebs buffer solution, plasma from normotensive and hypertensive dogs. The dose-response curve for NE was significantly (p < 0.05) increased when the mesenteric arteriole was perfused with the buffer solution containing 0.5 ml of plasma from hypertensive dogs per 100 ml of perfusate. There was no significant change with perfusion of plasma from normotensive dogs (0.5 ml of plasma per 100 m1 of perfusate). Figure 9 compares dose-response curve for NE obtained during perfusion of plasmas. Figure 8. 44 Typical response of the pressure of isolated mesentery artery to different doses of NE during perfusion of buffer solution (see method for detailed composition), plasma from chronic one- kidney perinephritis hypertensive (HPT Plasma) and normotensive dogs (NT Plasma). Plasma perfusion was 0.5 m1 of plasma per 100 ml of perfusate and flow rate was 2 ml/min. 3 9106;; “HMS ‘3}198 owsold ldH DWSD'd 1N 45 mm/Hg 09 j cor"- ooz’“> 09 —" 00I_* ooz“> 09 "I 001* ooz""'> oor‘" 0077’ A —09 '\ ,_.__' 'D —' OOl ‘— I "‘ 09I Figure 9. 46 Dose-response curve for NE in isolated mesenteric artery during perfusion of plasma from chronic one-kidney hypertensive (0-0) and normotensive (o-o) dogs. Plasma perfusion was 0.5 m1 of plasma per 100 ml of perfusate. Percent change in response above control is the percent difference between the rise in perfusion pressure caused by NE before and after the injection of plasma. Each point is the mean of 6 experiments. Vertical lines represent standard error of the mean. (*) Significantly higher (p < 0.05) than the effect obtained after the administration of plasma from normotensive dogs. 47 0—0 HPT Plasma o—o NT Plasma 100- _ _ O 0 8 6 AOKPZOU m>0m< mmZOammm wZ Z. may—44:0 FZmOm—md (n9) Figure9 48 4. Effect of plasma fractions on the activity of angiotensin II on blood pressure of ganglion-blocked and bilaterally nephrectomized rats We studied the effect of plasma fractions containing substances with small molecular weight on the pressor activity of All in Bioassay I. a. Plasma from one-kidney perinephritis hypertensive dogs was passed through Amicon Diaflo Ultramembrane 2 (UM-2 retainer fraction) and 05 (UM-05 retainer fraction). Substances retained by those membranes were tested in 8 rats in Bioassay I. The pressor activity of AII began to increase 5 to 10 minutes after the intravenous administration of both fractions, but the effect of UM-OS retainer was significantly higher (p < 0.05) as compared to the effect of UM-Z retainer (Fig. 10). Also, after the administration of UM-05 retainer fraction the rise in the response to AII was usually longer than after the administration of UM-2 retainer fraction. b. UM-OS retainer was further fractionated on gel filtration column (Bio-Gel P-2 1.5 x 90 cm) in order to determine the molecular weight of the humoral factor more closely. Figure 11 shows the ultraviolet absorbance at 230 nm of Bio-Gel P-2 fractions (close circles). Two pepetides, angiotensin II (M.W. 1100) and glycyl-glycine (M.W. 132), were used as molecular markers. It can be seen from Figure 11 that there were two main peaks; the substances represented 49 by the first peak (eluted volume between 102 to 148 m1) have molecular weights close to 1,000; and the substances represented by the second peak (eluted volume between 150 to 212) have molecular weights around 700 or 800. Fractions were freeze-dried and then redissolved in 0.9% NaCl, and bioassay was performed immediately after plasma fraction was passed through Dowex 50W-X2 resin to remove any angiotensin formed during the procedure or that may be in the plasma fraction. Fraction No. 20 (115 to 120 ml of eluted volume) from l-KHD produced a significant increase of the activity of AII in Bioassay I (Fig. 11. Open circles). Also, there was no significant increase in the bioassay response to AII after the administration of other fractions included in the first peak of eluted volume. These results were consistent with the previous step of the fractionation, which showed that substance(s) with molecular weight(s) close to 1,000 may be responsible for the effect of plasma from one-kidney hypertensive dogs and rats on the pressor response to AII and NE. Figure 12 shows the ultraviolet absorbance at 230 nm of Bio-Bel P-2 plasma fractions from normotensive dogs (close circles). The elution pattern was essentially the same as that obtained with plasma from hypertensive dogs. Fraction No. 20 from normotensive dogs produced a moderate increase in the response of Bioassay I to AII (Fig. 12. Open circles), but this effect was significantly lower (p < 0.05) 50 than that observed after the administration of fractionrwp. 20 from 1-KHD. Fifty ul of fraction No. 20 from 1-KHD raised the vasopressor activity of AII within the first five minutes after the administration of this fraction, and the effect always lasted more than 30 minutes. Fifty ul of fraction No. 20 from normotensive dogs did not produce significant changes in the response of the assay rat to AII (Fig. 13). These results suggest that in one-kidney Goldblatt type hypertension in rats and in one-kidney perinephritis hypertension in dogs a small molecular weight circulating substance may be responsible for an increased vasopressor response to AII and NE in those types of hypertension. Figure 10. 51 Effect of plasma fractions from chronic one- kidney perinephritis hypertensive dogs on AII vasopressor activity in bilaterally nephrecto- mized rats. Both Amicon Diaflo Ultrafiltration membrance UM-OS (0-0) and UM-Z (o-o) retainer fractions were assay in the same animals. Percent change in All response above control is the percent difference between rise in BP caused by AII before and after the injection of plasma fractions. Each point is the mean of four to eight experiments. Vertical lines represent standard error of the mean. (*) Significantly higher (p < 0.05) than the effect obtained after the administration of UM-2 retainer plasma fraction. 52 PERCENT CHANGE IN AII RESPONSE ABOVE CONTROL mo ho we no .5 OIOESOm DICE—SN 3 .A- . . q A m 3 .5 8 ._.__sm .35. ~32 33:0: maamamfimmoi 39.3 _0 3 q Nm wc Figure 11. 53 Effect of plasma fractions from chronic one- kidney perinephritis hypertensive dogs on AII vasopressor activity in bilaterally nephrecto- mized rats (o-o). Bioassays were performed immediately after fraction was passed through Dowex 50W-X2 resin column. Percent change in AII response above control is the percent difference between rise in BP caused by AII before and after the injection of plasma fraction. Each point is the mean of 5 experi- ments. Vertical lines represent the standard error of the mean. (*) Response was signifi- cantly higher (p < 0.05) than that obtained after the administration of plasma fraction from normotensive dog (see Fig. 11). Close circles (o—o) represent the ultraviolet absorbance of each fraction at 230 nm. Angiotensin II (M.W. 1,100) and glycyl-glycine (M.W. 132) were used as molecular weight markers. 54 0D230 Pm I oh I oh I o; ob :3. 233m 3 8 9.8 .8 woo P—H erfimU