Humana Press Humana Press M E T H O D S I N M O L E C U L A R M E D I C I N E TM Angiotensin Protocols Edited by Donna H. Wang Angiotensin Protocols Edited by Donna H. Wang Historical Perspective of the RAS 3 1 3 From: Methods in Molecular Medicine, vol. 51: Angiotensin Protocols Edited by: D. H. Wang © Humana Press Inc., Totowa, NJ Historical Perspective of the Renin–Angiotensin System John E. Hall “To understand a science it is necessary to know its history.” Auguste Comte, 1798–1857 The renin–angiotensin system (RAS) is now recognized as the body’s most powerful hormone system for controlling sodium balance, body fluid volumes, and arterial pressure. It is mainly for this reason that researchers continue to be fascinated with this system more than 100 years after its discovery. With the development of drugs that effectively block different components of the RAS, the therapeutic advantages of inhibiting this system in hypertension, heart fail- ure, diabetes, and other pathophysiological states have become apparent. Yet, as our knowledge of the physiology and molecular biology of this system con- tinues to accelerate, it is also obvious that there is still much to learn and that many talented scientists will continue to devote their careers to understanding how the RAS functions in health and disease. This brief review summarizes a few of the many milestones that have paved the way for our current understanding of the RAS. I have focused mainly on discoveries during the first 80 years and have not attempted to review the explosion of molecular biology literature during the past several years; much of this information can be found in other chapters of this volume. Other histori- cal perspectives of the RAS are presented in several excellent reviews (1–7). 1. Tigerstedt’s Discovery of Renin The birth of research on the RAS was in 1898 when Finnish physiologist Robert Tigerstedt of the Karolinska Institute in Stockholm and his student Per Gunnar Bergman published a landmark paper (8) entitled “Niere Uud Kreislauf” (“Kidney and Circulation”) in Skandinavisches Archiev für Physiologie. Their 4 Hall meticulous experiments documented the long-lasting pressor effects of renal cortex extracts and they named the substance renin (Fig. 1). Extracts of the renal medulla did not contain the active substance and transection of the spinal cord to remove the effects of sympathetic activation did not influence the pres- sor effects of renal extract injections, leading them to conclude that the blood pressure effects of renin depended upon stimulation of “peripheral vascular centres.” Tigerstedt further speculated that increased renin production might be important for the cardiac hypertrophy often observed in kidney diseases. It is not entirely clear why Tigerstedt decided to study the blood pressure effects of renal extracts, although work by Richard Bright in the 1830s pointed out the potential importance of renal disease as a cause of hypertension. Tigerstedt may also have been influenced by the French physiologist Brown- Sequard’s discovery of an adrenal hormone that stimulated the search for new hormones in research centers throughout the world. According to Mattias Aurell (4), another factor that may have led Tigerstedt to perform these experi- ments is that he “needed a paper” to present at the International Congress of Medicine in Moscow, Russia during summer 1897. This may explain why all 50 experiments described in Tigerstedt’s landmark paper were conducted Fig. 1. Tigerstedt and Bergman’s experiments demonstrating a pressor effect of crude saline extracts injected into four rabbits. Notice that the blood pressure response was biphasic, with a short-lived decrease and then an increase in blood pressure that was maximal at about two minutes. (Redrawn from data in ref. 8.) Historical Perspective of the RAS 5 between November 1896 and May 1897, with a few additional experiments conducted in autumn 1897. Tigerstedt did not continue his studies after he returned in 1901 to his native Finland where he became deeply involved in the administration of a new institute of physiology in Helsinki, and his discovery was not initially given much recognition. 2. The Goldblatt Experiments—A New Paradigm for Hypertension The period from Tigerstedt and Bergman’s discovery until the 1930s is often referred to as the “dormant period” for the RAS, although there were several unsuccessful attempts to verify their experiments. Also, Janeway (9)demonstrated in 1909 that ligation of one of the renal artery branches and removal of the contralateral kidney caused a 13–33 mmHg rise in blood pressure in three dogs. However, there was very little interest in renin until the 1930s when Harry Goldblatt and his colleagues of Case Western University in Cleveland, Ohio demonstrated in dogs that constriction of the renal arteries with silver clamps consistently produced chronic hypertension (10) (Fig. 2). This discovery was perhaps the greatest single advance in producing an experimental model of hypertension that resembled human hypertension. It also spawned a wave of studies examining the mechanisms of what became known as “Goldblatt hypertension” and renewed interest in the nature of the Fig. 2. Goldblatt’s experiment demonstrating that moderate constriction of the left renal artery (LK) followed by moderate constriction of the right renal artery (RK) cause sustained hypertension in a dog. (Redrawn from ref. 5.) 6 Hall pressor substance released by the kidney. As a pathologist, Goldblatt observed that hypertensive patients almost always had arteriosclerosis of the kidney. He reasoned that placing a clamp on the renal artery would mimic the ischemia produced by arteriosclerosis of renal blood vessels. Goldblatt was convinced that his dog models were paradigms not only of human renovascular hyperten- sion, but also of essential hypertension. In a series of elegant experiments sometimes forgotten by modern physiolo- gists, Goldblatt and his colleagues demonstrated that adrenalectomy, section of the renal nerves, spinal cord section, and other maneuvers designed to block the sympathetic nervous system did not significantly modify hypertension induced by renal artery stenosis. They also showed that constricting the aorta above the kidneys caused hypertension, whereas aortic constriction below the kidneys had little chronic effect on arterial pressure. Goldblatt’s experiments, which continue to be confirmed to the present day, paved the way for identify- ing the renal pressor system. The similarities of the pathologic changes pro- duced in Goldblatt’s experimental model with those found in human essential hypertension also led to the concept that the kidneys play a central role in the etiology of hypertension; this contrasted with the prevailing view that renovas- cular disease was primarily a consequence, rather than a cause of hypertension. Goldblatt did not initially consider renin to be a primary candidate in medi- ating hypertension caused by renal artery constriction, although he believed that a humoral substance was involved. Later experiments confirmed Goldblatt’s suspicion that the RAS was only a partial explanation of the mechanisms of Goldblatt hypertension. 3. Discovery of the Angiotensins The failure to demonstrate involvement of the nervous system in Goldblatt hypertension supported the concept that a humoral mechanism was involved, but as Goldblatt stated, the view that in the pathogenesis of hypertension due to renal ischemia, a humoral mechanism involving a hypothetical effective substance of renal origin plays a part of primary importance is based almost entirely upon indirect evidence. Thus, the search for the elusive pressor substance released from the kidney was renewed. Harrison et al. (11) and Prinzmetal and Friedman (12) in 1937 showed that saline extracts from ischemic kidneys of hypertensive animals raised blood pressure, although the results were sometimes inconsistent. Sev- eral other investigators also obtained negative results. In 1938, Juan Carlos Fasciolo, working on his doctoral thesis in the labora- tory of the great Argentine physiologist Bernardo Alberto Houssay, demon- strated the presence of a humoral pressor substance by grafting an ischemic Historical Perspective of the RAS 7 kidney to the neck circulation in dogs. In these experiments, the ischemic kid- ney released a substance that produced hypertension in the recipient dog (13). Further evidence that the ischemic kidneys from hypertensive dogs secreted a vasopressor substance came from the experiments of Houssay and Taquini in 1938 (14) who demonstrated that blood drawn from the renal vein of hyperten- sive dogs caused intense vascular constriction in the hind leg of toads. The idea that renin was not a constrictor itself, but rather acted as an enzyme to produce a pressor substance, was proposed in 1938 by Kohlsteadt et al. (15) working with Irving Page’s group in Indianapolis (and later at the Cleveland Clinic) and in 1939 by Mua-aoz et al. working with Braun-Menendez in Buenos Aires, Argentina (16). Thus, two laboratories separated by great distance embarked on a series of experiments to identify the pressor substance released from underperfused kidneys. Although the two groups followed different paths, they discovered almost simultaneously the substance in plasma that is now called angiotensin. Kohlstaedt, Helmer, and Page, while attempting to purify renin in 1938, found that the preparation had greater blood pressure effects when assayed in cats and dogs in vivo than in the isolated dog tail perfused with Ringer’s solution (15). Something in the blood appeared to enable renin to exert its pressor action, and when they added small amounts of blood to Ringer’s solution containing the purified renin, the solution elicited powerful vasoconstriction in the perfused dog tail. They gave the name “renin activator” to the putative plasma protein responsible for activation of renin. In 1940, Page and Helmer (17) isolated a pressor substance formed by the interaction of renin and renin activator and named it angiotonin. That same year Braun-Menendez et al. (18) found that the renal venous blood contained a substance that could be extracted in acetone, was thermostable, produced a sharp rise in blood pressure that lasted only 3–4 min, and they named this new substance hypertensin. It was soon recognized that hypertensin and angiotonin were the same substance, and for the next 18 yr, both terms were used, although hypertensin was probably more popular because it was chosen by Ciba Phar- maceuticals for their synthetic and commercially available angiotensin II prepa- ration. In 1958, Braun-Menendez and Page (19) agreed to name the pressor substance angiotensin; by extension the substrate from which angiotensin is released was called angiotensinogen. During the 1950s, there were amazing advances in the biochemistry of the RAS, with Leonard Skeggs and his colleagues at the Case Western Reserve University leading the way (see Table 1). Skeggs’ group purified angiotensin in 1954 and discovered that it existed in two different forms, a decapeptide called angiotensin I (Ang I), and an octapeptide called angiotensin II (Ang II), derived from the cleavage of a dipeptide from Ang I (20,21). In 1956, Skeggs’ group discovered angiotensin-converting enzyme (ACE) and published the 8 Hall amino acid sequence of the octapeptide Ang II (23). At about the same time, Elliott and Peart (24) in England also published the amino acid sequence of angiotensin. Skeggs’ group published papers in 1956 and 1957 that showed the active part of angiotensinogen is a 14-amino acid sequence (25). This period of rapid progress culminated in 1957 with the synthesis of Ang II by Bumpus et al. (26) at the Cleveland Clinic in the United States and by Rittel et al. (27) in Switzerland. The brilliant discoveries of the biochemical components of the RAS, the successful synthesis of Ang II, and the distribution of synthetic Ang II (Hyper- tensin) by Franz Gross and his colleagues at Ciba Pharmaceuticals paved the Table 1 Milestones In Renin–Angiotensin System (RAS) History 1898 Tigerstedt and Berman discover that an extract from rabbit kidney cortex increases blood pressure (ref. 8). 1934 Goldblatt induces hypertension in dogs by renal artery constriction (ref. 10). 1939–1940 Braun-Menendez et al. (refs. 16,18) and Page et al. (refs. 15,17) discover hypertensin and angiotonin (later known as angiotensin). 1954 Skeggs et al. purify angiotensin and discover that it exists in two forms, angiotensin I and II, and predict the presence of ACE (refs. 20,21). 1954 Elliot and Peart (ref. 24) and Skeggs et al. (ref. 23) report amino acid sequence of angiotensin II. 1956 Skeggs partially purifies renin substrate (angiotensinogen) and its N-terminal part (ref. 25). 1957 Bumpus et al. (ref. 26) and Rittel et al. (ref. 27) synthesize angiotensin II, which is widely distributed to researchers by Ciba Pharmaceuticals. 1958 Gross (ref. 28) articulates the hypothetical relationship between angio- tensin II and aldosterone. 1959–1961 Davis et al. (ref. 29), Laragh et al. (ref. 30) Genest et al. (ref. 31), and Ganong and Mulrow (ref. 32) demonstrate that angiotensin II regu- lates aldosterone secretion. 1968 Bradykinin-potentiating factor, described in 1965 by Ferreira (ref. 59) is shown by Bahkle (ref. 60) to inhibit the conversion of AI to AII. 1969 Radioimmunoassay methods for renin are introduced (refs. 53,54). 1969–1971 Peptide antagonists of angiotensin II are developed (refs. 55,56). 1971 Elucidation of structure and synthesis of bradykinin-potentiating peptide (teprotide) (ref. 61). 1977 Ondetti, Rubin, and Cushman describe a novel class of orally active ACE-inhibitors (captopril) (ref. 62). 1982–1988 Orally active, nonpeptide blockers of the AII type I receptor are described (refs. 63,64). Historical Perspective of the RAS 9 way for physiological studies of the RAS in the 1960s and 1970s. Also, the development of sensitive methods to measure plasma renin activity (PRA) and Ang II concentration in the late 1960s made it possible to examine the feed- back systems that regulate activity of the RAS and to differentiate between pharmacological and physiological actions of Ang II. 4. Role of Ang II in Control of Aldosterone Secretion For most of the 1930s until the 1950s, research on the RAS was directed toward its pathogenic significance in hypertension. In the 1950s Gross and colleagues began to investigate the physiological relationships between adre- nal hormones and the RAS in their laboratory in Heidelberg, Germany. Gross reviewed their observations in 1958, clearly articulating their finding that changes in sodium balance caused parallel changes in the RAS and aldosterone secretion and proposing that Ang II was an important regulator of aldosterone secretion (28). Although Gross did not have techniques available in his labora- tory to measure aldosterone secretion, he generously supplied synthetic Ang II (Hypertensin) to several researchers, including John Laragh, Jacques Genest, James O. Davis, and Francis Ganong and Patrick Mulrow who, although work- ing in different laboratories, proved almost simultaneously that Ang II was a powerful stimulator of aldosterone secretion. Between 1958 and 1961, Davis and colleagues demonstrated, with an elegant series of experiments in dogs, the presence of an unidentified aldosterone- stimulating hormone, that the source of the hormone was the kidney, and that infusion of synthetic Ang II stimulated aldosterone secretion (29). In 1960, Laragh and colleagues (30) showed that Ang II infusion raised plasma aldo- sterone in human volunteers and that same year Genest et al. (31) reported that Ang II increased urinary aldosterone excretion. Ganong and Mulrow (32) dem- onstrated in 1961 that an aldosterone-stimulating substance was released by the kidney during hemorrhage and confirmed that Ang II administration stimu- lated aldosterone secretion in dogs. These observations were the starting point for investigations of the physiology of the renin–angiotensin–aldosterone axis and the concept that blood pressure control by Ang II is closely interrelated to its renal sodium-retaining actions. Moreover, these studies provided a new focus on physiologic regulation of sodium and water balance and its major role in blood pressure regulation. Elucidation of Ang II’s role in regulating aldosterone secretion was pre- ceded by identification of the steroid structure of aldosterone in 1952 and development of physicochemical methods for its detection by Simpson and Tait (33); several other laboratories were busy developing reliable methods for measurement of aldosterone in urine with bioassay, chemical, and isotope methods. With improved methods to measure aldosterone in urine and plasma 10 Hall in the 1960s and with the development of specific Ang II antagonists and ACE inhibitors, the importance of Ang II in controlling aldosterone secretion was gradually revealed. 5. Role of Ang II in Controlling Renal Function One of the first studies to examine the role of the RAS in controlling renal function was in 1939 by Merrill et al. (34) who reported that injections of kid- ney cortex extracts decreased renal blood flow, increased blood pressure and urine excretion, and caused the kidneys to swell. He speculated that renin in the extracts caused contraction of the efferent glomerular vessels. This is in contrast to other pressor substances such as tyramine, which also decreased renal blood flow, but caused shrinkage of the kidney, decreased urine volume, and presumably contraction of afferent glomerular vessels. In 1940, Corcoran and Page (35) demonstrated in conscious dogs that iv infusion of the newly discovered angiotensin elicited essentially the same renal responses as observed with infusions of renal extracts—reduced renal blood flow and increased GFR and urine volume. With the widespread availability of synthetic Ang II, there were many stud- ies of the effects of Ang II infusion on kidney function in the 1960s, but their physiological relevance was uncertain because of the inability to accurately measure Ang II concentrations or to block the physiological actions of Ang II. A major advance toward understanding the physiology of the RAS came with the development of specific Ang II antagonists and ACE inhibitors. These new compounds allowed, for the first time, physiological and clinical studies aimed at the role of endogenous Ang II in controlling kidney function and arterial pressure. With the availability of effective blockers of the RAS in the early 1970s, it became apparent that even normal levels of Ang II have a tonic vaso- constrictor effect on the kidney. 5.1. The RAS and Macula Densa Feedback Control of GFR Goormaghtigh (36) speculated in 1937 that the juxtaglomerular cells of the afferent arterioles were the source of renin and that these cells might play a role in linking tubular and vascular functions of the kidney. This concept remained largely dormant until 1963 when Guyton (37) proposed, on the basis of a computer model, that the juxtaglomerular apparatus (JGA) played a major role in the feedback control of GFR. Less than a year later Thurau (38) pro- posed a similar idea and suggested that intrarenally formed Ang II was the mediator of this feedback; according to this hypothesis, increased sodium chlo- ride delivery to the macula densa stimulates renin release and causes constric- tion of afferent arterioles, thereby returning GFR toward normal. Although the existence of macula densa feedback control of GFR was later verified when Historical Perspective of the RAS 11 micropuncture methods became available, there were two aspects of this hypothesis that proved incorrect: (1) increased sodium chloride delivery was shown in 1964 by Vander and Miller (39) to decrease rather than increase renin secretion; and (2) Ang II was found to constrict the efferent arterioles to a greater extent than the afferent arterioles (40), an action that had been sug- gested by earlier studies of the renal effects of Ang II. 5.2. Blockade of Ang II Protects Against Hemodynamic Injury in Overperfused Nephrons When nephrons are overperfused and renin release is not appropriately sup- pressed, as occurs in certain types of renal disease or diabetes, blockade of Ang II formation, by decreasing efferent arteriolar resistance and glomerular hydrostatic pressure, protects the glomeruli from hemodynamic injury. Ander- son et al. (41) were the first to demonstrate in a series of experiments in rats with partial renal ablation that blockade of the RAS was more effective in slow- ing glomerular injury than other antihypertensive agents. Later studies in humans with diabetes and renal disease confirmed these early observations in rats. Although the beneficial effects of Ang II blockers and ACE inhibitors in slowing renal disease are now well established, the mechanisms involved are still debated. In vitro studies have shown than Ang II in high concentrations can promote vascular smooth muscle growth, increased collagen formation, and proliferation of extracellular matrix, particularly by mesangial cells. These findings have led to the hypothesis that blockade of the RAS protects the kid- ney from injury via nonhemodynamic mechanisms. However, an observation that is difficult to reconcile with this concept is that physiological activation of the RAS, by sodium depletion or renal artery stenosis, is not associated with vascular, glomerular, or tubulointerstitial injury as long as the kidney is not overperfused or exposed to high blood pressure. This suggests that the hemo- dynamic effects of Ang II, particularly efferent arteriolar constriction and increased arterial pressure, are necessary for many of the glomerular, tubular, and interstitial cell proliferative and phenotypic changes that occur when Ang II levels are excessively increased. Resolution of this controversy will await new experimental tools and novel approaches. 5.3. Intrarenal Actions of Ang II Control Sodium Excretion Early observations that acute infusions of large amounts of renin or kidney extracts often caused natriuresis and diuresis led to the notion that the main direct effects of Ang II on the kidney were vasoconstriction and a direct inhibi- tory effect on renal tubular sodium transport. When purified Ang II became widely available for studies in humans and experimental animals in the 1960s, Ang II was demonstrated to reduce sodium excretion by stimulating aldoster-