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CIO.‘ n )n. . o A!!! ... ‘ a a. la... ... .. s ....s. . \ u. . .. .wns .\ , ca?! s no.3 ...; ‘ .. a... i I; u.‘ t- .5 m: f u «I. cl! J { t o u 4“ a «at. o I I .‘ Nib... rkotm a“ ?\U -.. . 11“.... ..\ ~I .“r« I. s... RN- u.‘ . I. .. who. .ho ... _. z .- ... . . M I; m. c x 1.1.». ....-. .. . : . if u? y. . a , a . u -... . G. . -..: f . u I. \ II" I \“. I I\ . :8 u.-.“ . e.” . “:2. ,2 $6 . . u .. to .... ~ . \.. fl! \0 o ‘ . . S o. 1 Dirt .VIJI ”at ...l u :6 ‘00: II I fill.’ a F ...~ r . J t .... .. I- 35“ . ‘ ..-u: a . . . .f «JV . fl \ w ‘I I k -r ‘ b “ , “ L3,? L I B R /: K 1" Michigan Sta ta University ' . ‘3 tiC'C'c ABSTRACT THE EFFECT OF BILATERAL LIGATION OF THE CAUDAL RENAL PORTAL VEINS ON SOME CARDIOVASCULAR PARAMETERS IN THE SINGLE COMB WHITE LEGHORN ROOSTER BY Edward Arnold Cogger Experimental renal hypertension is produced in mam— malian species by partial constriction of the renal arteries. This presumably effects renin release from the juxtaglomerular cells by decreasing the blood pressure in the afferent arterioles and/or the sodium load to the macula densa. In birds, the renal portal veins, in addi- tion to the efferent arterioles, supply blood to the peri- tubular capillaries. Thus, ligation of the renal portal veins could effect the sodium load to the macula densa without decreasing the blood pressure in the afferent arterioles. In two experiments, bilateral ligation of the caudal renal portal veins in Single Comb White Leghorn (SCWL) roosters caused increases in mean blood pressure (MBP) of 7.8 percent and 6.8 percent. In the first experiment, the MBP increase was caused primarily by an increase in systolic blood pressure (SBP) from 181.4 mmHg to 210.9 mmHg. Edward.Arnold Cogger Diastolic blood pressure (DBP) was increased from 139.3 mmHg to 154.9 mmHg, but this difference was not statis- tically significant (0.15 > p > 0.10). In the second ex- periment the MBP increase was attributable to SBP increase (181.8 mmHg to 195.2 mmHg) and DBP increase (134.3 mmHg to 142.6 mmHg). Heart rate in the second experiment was significantly increased from 222.5 beats per minute to 299.0 beats per minute. There was a failure to detect renin release, as bio- assayed in nephrectomized rats, during in vitrg_incubation by kidney slices from SCWL roosters. This probably re- sulted from failure of avian renin to catalyze the conver- sion of rat plasma substrate to angiotensin. THE EFFECT OF BILATERAL LIGATION OF THE CAUDAL RENAL PORTAL VEINS ON SOME CARDIOVASCULAR PARAMETERS IN THE SINGLE COMB WHITE LEGHORN ROOSTER BY Edward Arnold Cogger A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Poultry Science 1970 LIST OF TABLES . INTRODUCTION . . OBJECTIVES. . . TABLE OF CONTENTS REVIEW OF LITERATURE . . . . . . . Experimental Renal Hypertension. . Blood Supply of the Chicken Kidney. PROCEDURES. . . Experiment 1 Experiment 2 Experiment 3 RESULTS. . . . Experiment 1 Experiment 2 Experiment 3 DISCUSSION. . . Experiments 1 and 2. . . . . . Experiment 3 SUMMARY AND CONCLUSIONS . . . . . . BIBLIOGRAPHY . . ii Page iii l4 14 16 l7 l9 19 22 26 28 28 31 33 35 LIST OF TABLES Table Page 1. Cardiovascular data for control, sham- operated, and bilateral caudal renal portal vein ligated SCWL roosters . . . . . . . 20 2. Summary of cardiovascular data from Table 1 represented as mean 1 standard error of the mean . . . . . . . . . . . . . . 21 3. Cardiovascular data measured prior to bi- lateral caudal renal portal vein ligation . . 23 4. Cardiovascular data measured after bilateral caudal renal portal vein ligation. . . . . 24 5. Differences in the cardiovascular data (Table 4 - Table 3) prior to and after bilateral caudal renal portal vein ligation. . . . . 25 6. Pressor response (mmHg) in nephrectomized male rats (220-225 grams of body weight) to a 0.2 m1 intravenous injection of Krebs-Ringer bicarbonate solution (KRB) incubated for 1 hour with SCWL rooster kidney slices. . . . 27 iii INTRODUCTION A considerable amount of scientific effort has been directed at the study of experimental renal hypertension and the relationship of the renin-angiotensin-aldosterone system to its pathogenesis. Primarily these studies have been conducted in mammalian species and only rarely have the submammalian vertebrates been the recipient of that effort. The chicken (Gallus domesticus) has seldom been the animal of choice in these areas of scientific research. Historically, a relationship between the kidney and cardiovascular disorders was first suggested in 1827 when Richard Bright reported a correlation between cardiac hypertrophy and chronic renal disease. Little progress occurred in determining the role of the kidney in the etiology of hypertension until 1898 when Tigerstedt and Bergman injected a crude renal extract intravenously and observed a sustained increase in blood pressure. They termed the pressor factor in this extract renin. Gold- blatt et_§l. (1934), in their classical experiments in which they partially ligated a renal artery in dogs, demon- strated that hypertension of renal origin could be pro- duced experimentally. Subsequently, experimental renal hypertension has been induced in a variety of laboratory animals including rats, rabbits, goats, and sheep (Pitts, 1963). Goldblatt's work triggered a renewed interest in the possibility that the kidney produced a pressor agent. This led to the rediscovery of renin by Pickering and Prinzmental (1938). Two groups of researchers (Page and Helmer, 1940; Braun-Menendez §E_§1,, 1940) discovered simultaneously that renin was an enzyme which acted upon a plasma substrate to produce a pressor agent. This pres- sor agent was called angiotensin. It was then shown that pressor activity was not caused by the decapeptide pro- duced from the plasma substrate by renin, but by an octa- peptide converted from the decapeptide (Skeggs et_al., 1955; Skeggs et_al,, 1956; Elliot and Peart, 1956, 1957). This conversion was thought to take place in transit through the lungs (Ng and Vane, 1968). The decapeptide and octapeptide were called angiotensin I and angiotensin II respectively. The ability of angiotensin II to stimu- late the secretion of aldosterone from the adrenal was shown by the work of Laragh et_al. (1960) and Genest et_al. (1960). The present work was undertaken to determine whether the chicken can be utilized in the study of experimental renal hypertension. fold: OBJECTIVES The specific objectives of this research were two- To produce experimental renal hypertension in the Single Comb White Leghorn male by bilateral ligation of the caudal renal portal veins, To determine if the Single Comb White Leghorn male kidney would release renin during an in yitrg incubation as assayed by the pressor re— sponse in neprectomized male rats. REVIEW OF LITERATURE Experimental Renal Hypertension The field of experimental renal hypertension had its beginning with the experiments of Goldblatt in 1934. Much interest was shown in Goldblatt's observations primarily because of their similarity to some forms of human hyper- tensive disease. Subsequently, great effort was devoted to the production and study of experimental renal hyper- tension in mammalian species primarily in dogs and rats. As a result of this effort, we are beginning to understand the role of the kidney in some forms of hypertensive dis- ease, and normal homeostasis of blood pressure and elec- trolyte balance. The production of a persistent experimental renal hypertension has been attempted by many methods. There are basically four methods of significance (Page and Corcoran, 1948). First, subtotal nephrectomy (partial surgical removal of the kidney) may result in hyperten- sion; however, the hypertension produced is irregular and complicated by renal insufficiency. Second, the ligation of major branches of the renal artery results in a hyper- tension similar to that produced by the above method. Third, the method developed by Goldblatt which involves the partial compression of the main renal artery by means 4 of a clamp. This procedure results in a very persistent hypertension in which renal excretory efficiency is not significantly impaired. Fourth, the kidney may be wrapped in a material such as silk or cellophane. The foreign body reaction produces a hull which results in a slowly developing but persistent hypertension. Some form of the third and fourth methods are most commonly used. By the use of Goldblatt's procedure, experimental renal hypertension has been produced in most laboratory mammals (Pitts, 1963). However, Lenel et_§1, (1948) re- ported a failure to produce hypertension in chickens after partial constriction of the renal arteries to one kidney and, in some birds, ligation of the contralateral ureter. No data or detailed description of procedure were pre- sented in this paper. The most obvious hemodynamic change in experimental renal hypertension has been the rise in blood pressure. The hypertension was characterized by an increase in systolic and diastolic blood pressure of equal magnitude (Page and Corcoran, 1948). In dogs (Grimson, 1939) and rats (Ledingham, 1966; Ledingham and Pelling, 1967) the average mean arterial pressure was increased significantly within 2 hours after a clamp was tightened onto the renal artery. The mean blood pressure in rats and dogs tended to plateau approximately 20 days after arterial constric- tion (Ledingham, 1966; Olmsted and Page, 1969). In the chronic stage of renal hypertension, the cardiac output was unchanged in dogs and increased 10% in rats; although, during the onset of the hypertension, cardiac output was decreased in both species (Olmsted and Page, 1965; Ledingham and Pelling, 1967). These ob- servations indicate that the rise in mean blood pressure was primarily due to an increase in peripheral resistance, i.e., vasoconstriction, since no important difference in the hematocrit was observed (Ledingham and Pelling, 1967). Plasma volume has been shown to increase in both rats and dogs after the onset of hypertension (Grollman and Shapiro, 1953; Ledingham, 1953; Ledingham and Cohen, 1964). The increase in plasma volume in rats accompanied the development of the hypertension but was not involved in its pathogenesis or necessary for its maintenance (Ledingham, 1953; Ledingham and Cohen, 1964). After the induction of renal hypertension, mean circulatory pres- sure which correlates with venous return has been observed to increase in dogs (Richardson et_al., 1964). The role of the renin-angiotensin-aldosterone system in experimental renal hypertension has been extensively reviewed by Page and McCubbin (1968). There are several reasons to suspect that the renin—angiotensin-aldosterone system has been involved in the pathogenesis of experi— mental renal hypertension. Renin activity, in the kidney and plasma, and angiotensin, in plasma, have sometimes been increased during hypertension. Also, the cessation of hypertension has occurred following the use of anti— renin; and the administration of renin or angiotensin has induced hypertension. Aldosterone has been shown to cause a hypertension which was enhanced by dietary salts; and aldosterone also intensified the pressor response to renin or angiotensin through its effect on sodium retention. The source of renin in the kidneys of mammals is the juxtaglomerular complex. This subject has been thoroughly reviewed by Smeby and Bumpus (1968). The juxtaglomerular complex is located at the vascular pole of the glomerulus and contains three types of cells: granular juxtaglomeru- lar cells (JG cells), agranular juxtaglomerular cells (lacis cells), and macula densa cells. The JG cells are found in the wall of the afferent arteriole and occasion- ally in the Polkissen (or polar cushion). The Polkissen, which is continuous with the efferent and afferent arteri- oles and lies between the distal tubule and the glomerulus, contains primarily lacis cells. Both the JG and lacis cell ultrastructure provide evidence for their relationship to smooth muscle cells. The macula densa cells are those cells of the distal tubule which are in juxtaposition to the Polkissen and the two arterioles. These cells are structurally different from other distal tubule cells. One difference is that the Golgi apparatus is on the basal Side of the nucleus, i.e., between the nucleus and the Polkissen. The juxtaglomerular complex is richly in- nervated by adrenergic nerves. Renin assays on micro- dissected renal tissue, correlation of renin content with JG granulation, immunofluoresence and organ culture have been used to show that renin was produced in the juxta- glomerular complex. Most investigators believe that renin is synthesized in the JG cells but some evidence suggests it may be synthesized in the macula densa. There are two theories for the control of renin re- lease (McCubbin, 1968). The "Baroreceptor Theory" postu— lates that the JG cells are stimulated directly by af- ferent arteriole blood pressure. The "Macula Densa Theory" states that the stimulus for renin release is the sodium load to the macula densa. JG cells have been reported in avian species. They have been demonstrated in the chicken and Japanese quail (Sokabe §E_al., 1969). There has been some disagreement as to the presence of macula densa cells. Sokabe et_§1. (1969) reported that macula densa cells were not present in the distal tubule of the chicken, whereas Edwards (1940) reported they were present. Capelli et_§1. (1970) ob- served the presence of a macula densa plaque in the domes— tic pigeon and the author of this thesis has observed the Same in the domestic turkey (unpublished) while conducting another research project. Renin or renin-like activity has been demonstrated in the kidneys of the chicken (Bean, 1942; Schaffenburg et_§1., 1960) and the domestic pigeon (Capelli §£;al,, 1970). Schaffenburg et_al, (1960) reported that chicken renin exhibited no activity when injected directly into trained dogs, but was highly active when incubated with homologous serum and then injected into dogs or when in— jected directly into chickens. Bean (1942) also demon- strated that chicken renin caused a pressor response in ducks and chickens but not in dogs, snakes, or toads. Ducks and chickens did not show a pressor response when kidney extract from toads, fish, pigs, sheep, dogs, or humans was injected. Chicken kidney extract, when incu- bated with chicken plasma, caused an increase in blood pressure when injected into dogs. When the extract was incubated with plasma from toads, dogs, oxen, or fish and subsequently injected into dogs, no increase in blood pressure occurred. Also, human, sheep, pig, ox, fish, or toad kidney extract elicited no pressor reSponse in dogs When incubated with chicken plasma (Bean, 1942). Capelli et a1. (1970) were able to demonstrate renin in the kid- neys of the pigeon by incubating the kidney extract with angiotensinase-free calf substrate and assaying in rats. Renin has been shown to be a proteolytic enzyme with a molecular weight of approximately 40,000 (Peart, 1969). Since a pure form of the enzyme has not been obtained, 10 direct measurement of renin was not possible; therefore, renin concentrations have been inferred from its ability to form angiotensin (Haber, 1969). Renin activity in the kidneys was most often measured either by the injection of extracts into rats (neprectomized and/or pentolinium- treated) or trained dogs, or by incubation of the extract with plasma substrate. The angiotensin formed was then bioassayed for its pressor activity using pure angiotensin II as a standard (Bunag and Masson, 1968). Plasma renin was determined by incubation procedures and assayed as above (Smeby and Bumpus, 1968) or by using isolated aortic strip preparations (Haber, 1969). Recently, DeJong (1969) has been able to measure changes in renin release of kidney slices incubated EE.XEE£2 and assayed in neprectomized rats. He reported that the amount released in a specified time period correlated well with plasma renin activity in normal, deoxycorticosterone acetate treated, and experi- mental renal hypertensive rats. All the present methods for measuring renin activity are subject to criticism be- cause very little is known about the catalytic environment required for maximum effectiveness of renin, the presence Of accelerators or inhibitors in the plasma, or conditions under which samples should be taken (Page et al., 1965). Blood Supply of the Chicken Kidney There are three renal arteries to each kidney of the domestic chicken. The anterior renal artery originates 11 from the aorta and supplies blood to the adrenals, testes or ovary, and the anterior divisions of the kidney. The middle and posterior renal arteries arise together from the sciatic artery and supply blood to the middle and posterior division of the kidney (Siller and Hindle, 1969). Sperber (1948) reported similar findings but also stated that one or more small arteries to the kidney originate from the femoral artery. Siller and Hindle (1969) were unable to confirm this finding. As the renal arteries ramify in the renal tissue their branches appear to follow the efferent veins; the intralobular arteries to each lobule originate from one or more of these branches (Sperber, 1948; Siller and Hindle, 1969). The intralobular arteries are arranged symmetrically around the central vein of the lobule. The intralobular artery gives off afferent arterioles that are directed toward the periphery of the lobule and break up into the capillaries of the glomeruli. The efferent arter- iole is also directed toward the periphery and enters the peritubular capillaries against the flow of the renal portal blood (Sperber, 1948; Siller and Hindle, 1969). The kidney of birds has a renal portal system. The renal portal system supplies venous blood to the anterior, middle, and posterior divisions of the kidney and its Capability of functioning has been demonstrated (Sperber, 1946, 1948, 1960). The caudal and cranial renal portal Veins originate from the external iliac vein (femoral vein) 12 between the point where it enters the body cavity and the renal portal valve. They supply the middle and posterior lobes, and anterior lobe, respectively (Sperber, 1948; Akester, 1964). The cranial renal portal vein was shown to be continuous with the vertebral venous sinuses (Akester, 1967) and to give rise to the interlobular veins of the kidney's anterior division (Sperber, 1948). The caudal renal portal vein courses caudally through the renal tissue. The sciatic vein joins it between the middle and posterior divisions of the kidney. The caudal renal portal veins form an anastomoses with the hypogastric veins from the tail region and the coccygeomesenteric vein (Sperber, 1948; Akester, 1964). The caudal renal portal vein gives rise to the interlobular veins of the kidney's middle and posterior division (Sperber, 1948). The interlobular veins are continuous with the peritubular capillaries which are drained by the intralobular or central vein (Sperber, 1948; Akester, 1964; Siller ahd Hindle, 1969). The intralobular veins leave the lobule and eventually drain into the anterior and posterior renal veins. The renal veins form an anastomoses with the external iliac vein just proximal from the renal portal valve and the large vein formed unites with the vein from the contralateral side to form the posterior vena cava (Sperber, 1948; Akester, 1964). The renal portal valve is located within the lumen of the external iliac vein proximal to the origin of the l3 crannial renal portal vein and distal to the anastomosis between the external iliac and renal veins. This valve contains smooth muscle and autonomic nerve fibers (Gilbert, 1961; Akester and Mann, 1969). The location of the renal portal valve suggests that it can control the flow of venous blood such that it flows to the kidney or bypasses the kidney to the heart. Physiological evidence for this possibiltiy was suggested by Sperber (1948), Rennick and Gandia (1954), and Akester (1964, 1967). PROCEDURES The research reported in this thesis was divided into three experiments. Experiments 1 and 2 were con- cerned with the production of experimental renal hyper- tension and Experiment 3 with in vitro release of renin by avian kidney slices. Experiment 1 This experiment was designed to determine the effect of bilateral caudal renal portal vein ligation on the following cardiovascular parameters: heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and pulse pressure (PP). SBP and DBP were recorded on a Grass Model 5 polygraph using a Statham P 23A arterial pressure transducer. Blood pressure was measured by cannulation of the left carotid artery close to the bifurcation into the left internal and external carotid arteries. A general anesthetic was not used. Local anesthesia was produced by the subcutane- ous injection of procaine. HR was determined from the recording. PP and MBP are derived quantities and were Calculated as follows: PP = SBP - DBP, MBP = DBP + 3/8 PP. l4 15 The experimental design consisted of three treatments with five Single Combed White Leghorn (SCWL) roosters per treatment. The data was subjected to one-way analysis of variance (ANOVA) to test HO: “1 = HZ = U3 for each of the above parameters (HR, SBP, DBP, PP, MBP). Supplemental statistical analyses were conducted. Duncan's New Multiple Range (Steel and Torrie, 1960) test was used to test HO: “1 = pi, for all possible pairs of means. The test of HO: of = a: = 0% was determined by Cochran's test (Kirk, 1968).* The control birds (Treatment 1) were subjected to no experimental procedure other than the measurement of blood pressure. These measurements were interspersed with the measurement of blood pressure in Treatments II and III. In Treatments II and III a general anesthesia, Nembutal (sodium pentabarbitol), was administered until the bird reached a surgical depth (approximately 30mg of Nembutal/kg of body weight). A dorsoventral incision was made through the skin just caudad to the last rib on both *The best estimate for the standard error of the mean (SX ) when equality of variance has been established is given by the following: 5' = MEE where SX = Standard error of the mean for Xi n1 Xi .th the 1 treatment. MSE = Error mean square ni = number of observations in the ith treatment. Therefore, when the number of observations per treatment are equal the standard error of the means are also equal (Steel and Torrie, 1960). 16 the right and left sides. The abdominal muscles were separated and the abdominal air sac was punctured. The intestines were gently pushed aside to expose the ventral surface of the kidney. A hemostat was attached to the caudal renal portal vein close to its origin from the external iliac vein. In Treatment II the hemostat was sub- sequently removed and the incision closed (sham-operated). In Treatment III a small hemostasis clip was used to ligate the vein adjacent to the hemostat. Then the incision was closed. Blood pressure of the birds in Treatments II and III was measured, ten days postoperatively. Experiment 2 This study was similar in purpose to Experiment 1. In this experiment each animal was its own control. Again, the test animals used were SCWL roosters. Prior to sur- gery, HR, SBP. DBP, MBP, and PP were determined as des- cribed in Experiment 1. The roosters were then subjected to the same surgery as Treatment III in the first experi— ment. Ten days postoperatively the right carotid artery was cannulated and the HR, SBP, DBP, MBP, and PP were determined. Student's t test for paired observations was used for the statistical analyses of data (Steel and Torrie, 1960). The data from the previous experiment and evidence in mammalian species that a decrease in renal plasma flow 17 caused an increase in blood pressure warranted a one- tailed test of H : u = 0. 0 xi — Xi' Experiment 3 This experiment was designed to determine if the kidneys of SCWL roosters released renin in yitrg and if a dose-response relationship existed. This study employed the use of three SCWL roosters. Each bird was killed by cervical dislocation and its kidneys were immediately removed and placed on ice. The kidneys were then cut into slices (8-12 slices per 100 mg of tissue). The kidney tissue of each bird was divided into three quantities (200 mg, 350 mg, and 500 mg portions) for in XEEEE incubation. The avian kidney slices were in- cubated, with some modifications, according to the pro- cedures developed by DeJong (1969) for rat kidney slices. A Dubnoff metabolic incubator was used for the incubation. The kidney slices were placed in 20 ml beakers with 2 ml of Krebs-Ringer Bicarbonate (KRB) solution (with 260 mg glucose per 100 ml KRB) and were then pre-incubated for 15 minutes at 41° C under an atmosphere of 95% O2 - 5% CO2 (6 c.f.h. at 70° F and 14.7 psi). The KRB was poured off and 2 m1 of fresh KRB added. The kidney slices were incu- bated for one hour. At the end of the incubation period the KRB was poured into stoppered vials and held at room temperature. Within 3 hours each sample was bioassayed by the pressor response after a single 0.2 ml injection of the l8 incubated KRB into a urethane anesthetized (125-150 mg/lOO mg of body wt.) Sprague-Dawley male rat (220-225 gm) which had been nephrectomized 16-20 hours previously. Nephrectomy was performed under ether anesthesia and carotid blood pressure was recorded with Grass Model 7 polygraph using a Statham P23DC transducer. The data were to be analyzed statistically by simple linear regression (Steel and Torrie, 1960); however, no statistical analysis was necessary because it was clear that no meaningful regression line existed. RESULTS Experiment 1 The data collected in the first experiment is tabu- lated in Table 1 and summarized as means and standard errors in Table 2. Analysis by Cochran's test indicated that variances between treatments for the different param- eters were equal. In the birds with bilateral ligation of the caudal renal portal veins an average systolic blood pressure (SBP) of 210.9 mmHg was observed and this was significantly (p < 0.05) higher than 188.7 mmHg and 181.4 mmHg observed in the control and sham-Operated birds. Also, in the ligated birds the pulse pressure (PP) of 56.0 mmHg was significantly (p < 0.05) greater than the PP's of 40.5 mmHg and 42.1 mmHg in the control and sham- operated birds. The mean blood pressure (MBP) in the ligated birds (175.9 mmHg) was significantly (p < 0.05) elevated above the sham-operated (155.1 mmHg) but not the control (163.1 mmHg) birds. No differences in heart rate (HR) and diastolic blood pressure (DBP) were detected between the treatments. 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In this experi- ment each animal was used as its own control since the data from the first experiment indicated that the sham-operated birds were not significantly different from control birds. Tables 3 and 4 contain the data collected before and ten days post-operative to ligating the caudal renal portal veins; whereas, Table 5 shows the change that was observed after bilateral ligation represented as a difference, i.e., change = postoperative value - preoperative value. Substantial increases in HR were observed in all birds except bird 6. SBP, DBP and MBP were increased by the surgical procedure in birds 1, 2, 4, 5, and 6. It was an interesting observation that bird 3 in which SBP, DBP, and MBP decreased by 11.3 mmHg, 6.5 mmHg, and 8.3 mmHg respectively also had the greatest increase in HR (up 104 beats/minute) and all measures of the blood pressure (SBP = 208.8 mmHg, DBP = 144.5 mmHg, and MBP = 168.6 mmHg) were initially the highest in the group. Pulse pressure was dramatically changed in only birds 2 and 6. HR which was up 76.5 beats per minute was signifi- cantly increased (p < 0.01) over preoperative levels. SBP, IIBP, and MBP were elevated significantly (p < 0.05) over Ixreoperative levels by 13.5 mmHg, 8.3 mmHg and 10.3 mmHg, 23 Imm.¢ mfi.m Iow.¢ Iso.m Imo.ma .m.m + m.s¢ + H.mmH + m.va + m.HmH + m.mmm qmmz o.Hv e.mmH o.mHH o.¢ma o.Hmm w m.Hm m.oeH «.mma m.omH o.osm m o.mm m.mma «.mwa m.mmH o.«mm e m.¢m m.mma m.¢qa m.mom o.mma m o.me w.mmH m.mma m.HmH o.mmH m m.mv m.sma ~.mmH m.sma o.oam H Ammssv mm images mm: Ammssv mma Ammssv mmm A.:Hs\mummnv mm .02 wnflm .aOHummHH cflm> kuuom Hmcmn Hmcsmo Hmnmnmafln ou Howum cmusmmme mnmp swasomm>oflcumonl.m mqmfie 24 Ims.m ms.m mm.m Inm.v Imo.mH .m.m + o.mm m.mmH o.mea + ~.mma + o.mmm cam: m.mm m.meH m.s~H o.mmH o.mmm m m.vm e.sma m.vea m.msH o.m¢m m v.5m m.msa m.mma ~.HH~ o.qu a m.mm m.omH o.mma m.sma 0.0mm m m.mm v.soa m.mva m.mom o.mmm m m.mm m.mma m.msa m.mma o.mom H Ramses mm homage mm: Ammssc mmo Ammesv mmm A.qns\mummnv mm .02 vunm .coflummfla QH®> HMHHOQ HMQOH HMMVDQO HMHO#MHHQ Hwflmm Uwhflmmwg M¥MU HMHSUWM>OflUHMUII.v WQMANH. 25 .Hm>mH wH on“ um OHmN cmcu prmmhm mHucmonwcmHm mH cmmzss .Hm>mH mm mcu um OHmN smsg HmummHm mHucmoHMHcmHm mH nmmz* Ins.m who.v wmm.m on.m .wms.HH I .m.m + m.m + H.0H + m.m + m.mH + m.ms + cams m.sH m.mH m.vH o.m~ o.Hm o o.m m.oH m.mH m.mH o.oo m v.m m.HH G.OH o.mH 0.0m H m.H- m.m- m.m- m.HH- o.HOH m m.OH o.HH o.OH m.om 0.0m N o.m m.s 0.8 o.HH o.mm H Homage mm Homage mm: Hmmssc mmo Homage mmm H.2Hs\mpmwnv mm .02 wuHm .coaummfla cflm> Hmunom Hmsmu Hmcsmo HmnmuMHHn Hmpmm cam on HOHHQ Am anme I v manmev mumc HMHdomm>OH©Hmo mcu CH mmocmHmMMHonu.m mqmfle 26 respectively. An average increase in PP of 5.2 mmHg was not found to be significant. Experiment 3 As outlined in the procedures section the kidneys from three SCWL males were to be used. As the data in Table 6 shows there was no pressor response elicited at any of the three quantities of tissue incubated from the first two birds; therefore, the experiment was terminated after the second bird was bioassayed. The results (Table 6) show that no renin activity was present at any of the quantities of avian kidney tissue incubated. No statisti— cal analysis was done because it was clear that no mean- ingful regression line existed. 27 TABLE 6.--Pressor response (mmHg) in nephrectomized male rats (220-225 grams of body weight) to a 0.2 ml intravenous injection of Krebs-Ringer bicarbonate solution (KRB) incu- bated for 1 hour with SCWL rooster kidney slices. Mg kidney slices/2 m1 KRB Bird No. 200 350. 500 l 0 mmHg 0 mmHg 0 mmHg 2 0 mmHg 0 mmHg 0 mmHg Tr.“ ”...”.w.‘ "'f-“-§"""‘ 1'. k1“. DISCUSSION Experiments 1 and 2 Normal blood pressure and heart rate characteristics of the rooster were described by Sturkie (1965) to be as 3} follows; HR = 300 beats/minute, SBP = 190 mmHg, and DBP = (:4_ 150 mmHg. The control roosters in Experiment 1 very closely ~ approximate those values whereas those in Experiment 2 show E some major differences. The birds in Experiment 2 as a group showed a marked bradycardia. Since cardiac output is determined by the heart rate and stroke volume one might suspect that the slight hypotension observed was a result of decreased cardiac output, but the data in Table 3 show that the birds (5 and 6) with the lowest blood pressure have the highest heart rate and that the bird (3) with the highest blood pressure has the lowest heart rate. There- fore, it would appear that the bradycardia and hypotension reflect a random variation of these parameters in a normal population. The results from Experiment 1 (Table 2) indicate an increase in SBP, MBP, and PP; however, DBP does not appear to be increased significantly. Page and Corcoran (1948) stated that experimental renal hypertension was charac- terized by proportional increases in SBP and DBP and no change in cardiac output; therefore, it was a result of 28 29 increased resistance to flow. If cardiac output increased in the absence of a peripheral resistance change, there would be an increase in SBP and PP but not DBP. The data suggest that the cardiac output may have been increased with little or no change in peripheral resistance. It is entirely possible that ligation of the caudal renal portal veins could increase cardiac output by eliminating a major renal portal shunt away from the heart (Akester, 1967) and increasing venous return through the renal portal valve to the heart. In fact, increasing a cardiac output by increas- ing venous return was one of the functions which has been suggested for the renal portal valve (Gilbert, 1961). In Experiment 2, HR, SBP, and DBP were significantly increased but PP was not significantly increased. This suggests that the increase in blood pressure was the result of increased peripheral resistance with little or no changes in cardiac output in spite of the large increase in HR. This was similar to the hypertension reported by various authors (Page and Corcoran, 1948; Olmsted and Page, 1965; Ledingham, 1966; Ledingham and Pelling, 1967); however, Olmsted and Page (1965) reported no change in HR in hyper— tensive dogs, and Ledingham and Pelling (1967) reported an increase in cardiac output roughly attributable to equal increases in stroke volume and heart rate in hypertensive rats. 1 ‘- r—4 .‘m‘_ 1‘ ...-ca..- . 1“— -.— .... 1 " R‘- an: 3O Diastolic blood pressure is increased significantly in Experiment 2 but not Experiment 1. This difference led the author to speculate two different origins of the hyper- tension produced in these experiments. This is a peculiar conclusion to reach since there were virtually no differ- ences in experimental procedures on either group of birds. ' 3.3.", w- - ud— The percentage increase in DBP in Experiment 1 was greater ...o. when using sham-operated birds (11.2%) as a basal level and less when using control birds (4.5%) as a basal level than in Experiment 2 (6.2%). Since Experiments 1 and 2 have trill—3 "xii ‘u- _ 8 different designs it is not possible to pool the data, but by use of chi-square test it is possible to pool the prob- abilities to test the null hypothesis (Steel and Torrie, 1960) that bilateral ligation of the caudal renal portal veins causes no effect on DBP. This test indicates that DBP is significantly increased (p < 0.05) for both experi- ments. Therefore, it is concluded that the hypertension in both experiments is similar. There was one major discrepancy in both groups of hypertensive birds as compared to mammalian experimental renal hypertension. The increased MBP in Experiment 1 was 13.4 and 7.8 percent and in Experiment 2 6.8 percent. Rats (Ledingham, 1966) showed a mean increase in MBP at 10 days of 25 percent and dogs (Olmsted and Page, 1965) 77 percent. This difference might have been partly attributable to the higher initial blood pressure in birds. Also, this in. 31 difference may have resulted by decreasing the sodium load to the macula densa stimulating renin release without the concommitant decrease in afferent arteriole blood pressure which may potentiate that stimulus. Experiment 3 There are at least five explanations for the data . :7 ‘— . reported in Table 6, which indicate renin activity was not ! present in the KRB after incubation with avian kidney slices. First, the kidney of the SCWL rooster may not pro- duce renin, although this would appear to be unlikely since r ’5‘!“ l IA-ka-“mu‘ w I ‘0 ! l." ( Luv) ..2. its presence has been reported in chickens (Bean, 1942; Schaffenburg, 1960) and pigeons (Capelli, §E_gl., 1970). Second, the avian kidney slices may not have released renin in yitrg under the same conditions as rat kidney slices. The temperature of the bath was adjusted to 41° C instead of 37° C because this more closely approximates the body temperature of chickens; still this explanation remains a possibility. Third, renin may be released by the avian kidney but destroyed or inactivated. Renin was reported to be very stable (Page and McCubbin, 1968); but, as a pre- caution the KRB was assayed within 3 hours after incuba- tion. Fourth, renin may be released but not in sufficient quantities to be detected by this assay procedure. DeJong (1969) using rat kidney slices, incubated 110 mg of tissue; however, in this study up to 500 mg of kidney tissue from 32 the chicken was incubated and still zero response was ob— served. Fifth, the most probable explanation would appear to be that avian renin was not able to convert rat angio- tensiongen to angiotensin I in 3139, Species specificity has been well documented for renin. Schaffenburg et_al. (1960) and Bean (1942) reported no response when chicken kidney extracts were injected into dogs; however, Capelli et_al, (1970) demonstrated that avian renin from pigeons when incubated with calf substrate did produce angiotensin when assayed in rats. To the author's knowledge, no one has yet reported an absolute specificity between the chicken and rat. SUMMARY AND CONCLUSIONS Bilateral caudal renal portal vein ligation in SCWL roosters caused, 10 days postoperatively, a hypertension in two different experiments. In the first experiment a systolic hypertension was produced, whereas in the second experiment, a diastolic hypertension and tachycardia were produced. When both experiments are considered together significant increases in systolic, diastolic, and mean blood pressure are produced by the surgical treatment. The percentage increase in mean blood pressure was con- siderably less than that observed in rats and dogs with experimental renal hypertension. The hypertension pro- duced by bilateral ligation of the caudal renal portal veins appears to be similar to mammalian experimental renal hypertension in direction of blood pressure changes, but not in magnitude. This may have resulted from higher initial blood pressure and/or the afferent arteriole pres- sure stimulus counteracting the sodium load stimulus for renin release. Renin release was not demonstrated during in yitrg incubation by avian kidney slices as assayed in nephrecto- Inized rats. This was probably the result of the failure of avian renin to catalyze the production of angiotension from rat plasma substrate (angiotensin). 33 34 Further studies need to be conducted to confirm the results of this research, to determine the effect of bilateral caudal renal portal vein ligation on sodium and potassium excretion, and to determine the role of the renin— angiotensin-aldosterone system, in birds, on electrolyte homeostasis. TRIP..- -.‘ «T t‘TT—._'. ‘ “TE?" L. .... BIBLIOGRAPHY Akester, A. R., 1964. Radiographic studies of the renal portal system in the domestic fowl (Gallus domesti— cus). J. Anat. Lond. 98 (3): 365. Akester, A. R., 1967. Renal portal shunts in the kidney of the domestic fowl. J. Anat. 101 (3): 569. Bean, J. W., 1942. Specificity in the renin—hypertensinogen reaction. Am. J. Physiol. 136: 731. Braun-Menéndez, E., J. C. Fasciolo, L. F. Leloir, and J. M. Monoz, 1940. The substance causing renal hypertension. J. Physiol. 98: 283. Bright, R., 1827. 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