Angiotensin Protocols [Methods in Molec Med 51] - D. Wang (Humana) WW

486 Pages·2020·4.58 MB·English

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
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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

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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.)

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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
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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

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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
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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

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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
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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-