Clinical Science (1970) 38,157-174.
THE RELEASE OF RENIN INTO THE RENAL
CIRCULATION OF THE ANAESTHETIZED DOG
K. F. H O S I E , J. J. BROWN, A. M. H A R P E R , A. F. LEVER,
R. F. M A C A D A M , J. M A c G R E G O R A N D J. I. S. ROBERTSON
MRC Blood Pressure Unit, and Department of Pathology, Western Infirmary, and
Department of Veterinary Physiology and Wellcome Surgical Research Laboratory,
University of Glasgow
(Received 18 April 1969)
SUMMARY
1. In anaesthetized dogs the rate at which renin was released into the circulation of
the right and left kidneys was estimated from renal blood flow, haematocrit, and the
V-A renin concentration difference across the kidney. Renin was also measured in
samples of renal lymph collected at the same time.
2. The effect on renin release of reducing blood flow in one kidney was studied.
For all observations (control and experimental), renal venous plasma renin concentration (RVR) was directly related to arterial plasma renin concentration and to
renin release; RVR was inversely related to renal plasma flow.
3. The concentration of renin in renal lymph was considerably higher than that in
renal venous plasma taken at the same time. Arterial plasma renin concentration was
directly related to the sum of the rates at which renin was released from the two
kidneys.
4. Clamping the renal artery of one kidney for 1 hr led to a marked reduction of
renal blood flow, to a marked increase in RVR and to a variable change in renin
release. Removal of the clamp was followed by increased renin release and by reversal
of a previously positive V-A renin difference in the control kidney.
5. On several other occasions negative V-A renin differences were observed. That
is, more renin appeared to enter the kidney in arterial blood than left in the renal
vein.
Two groups of observations suggest that renin is released from the kidney into blood: firstly,
the concentration or activity of renin in renal venous plasma is generally higher than that in
either arterial or peripheral venous plasma (Skinner, McCubbin & Page, 1964; Judson &
Helmer, 1964; Vander & Miller, 1964; Brown et al., 1966; Kaneko et al., 1967; Nash et al.,
1968) and secondly, removal of both kidneys is followed by a prompt decrease in the activity or
concentration of renin in plasma (Gross, Regoli & Schaechtelin, 1963; Lever & Robertson,
Correspondence: Dr A. F. Lever, MRC Blood Pressure Unit, Western Infirmary.Glasgow.
157
158
K . I;. Hosie et al.
1964; Toussaint et al., 1966; Devaux et al., 1968; Brown et al., 1969). Measurement of renin
release is complicated by the fact that renin leaves the kidney by three routes: blood, lymph
(Lever & Peart, 1962) and urine (Brown et al., 1964a). Further problems arise from the limited
precision of renin assay techniques (see Discussion).
The object of the present experiments was to develop a technique for measuring the rate at
which renin is released into the renal circulation of the anaesthetized dog and to test this
technique in circumstances in which renal blood flow is grossly reduced. A preliminary report
of some of this work has been published previously (Brown et al., 1968).
METHODS
Terminology and calculation of renin release
By ‘renin release’ we mean the net rate at which renin is added to blood during its circulation
through the kidney. This does not include renin leaving the kidney in lymph and urine. The
rate of renin release has been calculated using the Fick principle:
(Renal venous plasma renin concentration - Arterial plasma renin concentration) x
Renal plasma flow
No allowance has been made for the small increase of plasma renin concentration which
would have been produced within the kidney by the removal of water during the formation of
lymph and urine. Renin release as defined here is a balance between renin which enters the
kidney in arterial blood and renin which leaves the kidney in the renal vein. Such a net rate of
release should be distinguished from the gross rate at which renin is liberated from the juxtaglomerular apparatus into blood. If arterial and renal venous plasma renin values are identical
(as in some instances illustrated in Fig. 3), net renin release is zero. Renin may nevertheless be
liberated from the juxtaglomerular apparatus at this time and an identical amount of renin
may be removed from renal blood, thereafter being inactivated, removed in lymph or excreted
in urine. In these circumstances, the rate at which renin is liberated from the juxtaglomerular
apparatus is positive at a time when the rate of net renin release is zero.
Surgical techniques
Dogs weighing 18-6-40kg were anaesthetized either with intravenous pentobarbitone sodium
(25 mg/kg) and chloralose (50 mg/kg) or with thiopentone sodium (25 mg/kg) and nitrous
oxide (75% N,O; 25% 0,).
Intermittent positive pressure ventilation was maintained throughout the experiment via a
cuffed endotrachael tube. Both femoral arteries, one femoral vein and both external jugular
veins were cannulated with polyethylene tubing containing saline and heparin (2500 i.u.
heparin/100 ml of 0.9% NaC1). The kidneys were then exposed through loin incisions and the
dog was suspended prone in a special frame (Hosie, 1969) which provided good exposure of
both kidneys at the same time. Both ureters were catheterized and, during a period of renal
artery occlusion which lasted on average for 4.4 min, the renal veins were cannulated and
renal blood flow was diverted through polyvinyl tubing of 4-6 mm internal diameter to the
jugular vein on the same side. The animal was given 2500 i.u. of heparin intravenously at this
stage. .
Renin release
159
Measurements
Blood flow was measured in the external venous circuit using a bubble flowmeter, the
calibration is shown in Fig. 1. Renal plasma flow was derived from renal blood flow and the
mean of duplicate measurements of haematocrit. Plasma renin concentration (Brown et al.,
1964b) was measured in 15-20 ml blood samples withdrawn simultaneously from both venous
circuits and from a catheter in the abdominal aorta. A volume of Dextran (5%) +saline (0.9%)
equal to that of the withdrawn blood was injected intravenously after each batch of samples
had been taken. 0.9% NaCl solution was infused into a femoral vein at a rate (0.02-0.35 ml
kg-I min-') which depended upon urine flow and the loss of blood during surgery. Mean
systemic arterial pressure was measured continuously via a catheter in the other femoral
artery using a damped mercury manometer. Osmolality and volume were measured in halfhourly urine samples from both kidneys.
1'
I
I
I
I
I
I
100
200
300
Blood flow (actual) (ml/rnin)
I
I
400
FIG. 1. Calibration of bubble flow meter: measuring volume 30.0 ml, bore 4.0 mm, measuring
length 235 cm. Calculated flow was derived from bubble transit time and measuring volume.
Measured flow was obtained using a graduated cylinder. Points and bars represent the mean and
SD of six to ten observations.
Renal lymph
Renal lymphatics were cannulated as previously (Lever & Peart, 1962), and eighteen 30
min collections of lymph were made from four kidneys in three animals. The lymph was
stored frozen. Lymphatic renin was measured by incubating 1 ml of dilute lymph (0.1 ml of
renal lymph diluted with 0-9 ml of 0.15 M, pH 5.7, phosphate-saline buffer) with 4 ml of
standard ox-substrate. The concentration of renin in this incubation mixture was estimated as
previously (Brown et al., 1964b) by measuring the rate of angiotensin formation and by reference of this value to a calibration curve relating the amount of standard dog renin in an
K. F. Hosie et al.
160
incubation mixture to the velocity of angiotensin formation (Fig. 2). As in the earlier method, a
unit of renin is the amount which produces angiotensin at a velocity of 0-1 pg ml-' hr-'.
Lymph was not extracted in any way prior to assay.
The angiotensinase content of lymph was tested in triplicate by incubating 0.1 ml of a stock
sample of dog lymph (derived from three animals) with 4.9 ml of a solution of angiotensin in
phosphate-saline buffer (0.1 pg/ml). The survival of angiotensin was measured after incubation for 24 hr.
The ability of renin substrate present in lymph (Friedman, Marx & Lindner, 1943; Lever &
Peart, 1962) to interfere with the assay was tested by incubating 0.1 ml samples of lymph with
4.9 ml of phosphate-saline buffer for 48 hr. The velocity at which these incubation mixtures
produced angiotensin was compared with that of 0.1 ml of the same lymph incubated with
0.9 ml of phosphate-saline buffer and 4 ml of ox-substrate.
0 0002
t
I
0.0 I
I
01
I
10
Dilutlon
FIG.2. Calibrationcurve using standard dog renin with comparison of the effect on the velocity of
angiotensin formation of serial dilution of lymph, and of two dog plasma extracts. 0, Standard
dog renin; 0 , renal lymph; A , dog plasma extract I ; x ,dog plasma extract 11. The lowest group
of three filled circles was without added renin.
Renal lymph was tested for inhibitors or accelerators of the renin-substrate reaction by its
effect on a standard dog renin, ox-substrate reaction (see Table 1 for volumes used).
The quantitative characteristics of the lymph assay technique were tested by comparing the
effect on the velocity of angiotensin formation of serial dilution of lymph and standard dog
renin. The dilutions used are shown in Fig. 2.
Experimental procedure
Each experiment was divided into periods of 30 min (see Fig. 8). At the mid-point of each
period, renal blood flow was measured and samples were taken for determination of haemato-
Renin release
161
crit, right and left renal venous plasma renin concentration and arterial plasma renin concentration. Renal blood flow was otherwise measured at approximately 3-min intervals throughout
the experiment. Arterial pressure was recorded continuously and the half-hourly urine collections lasted from the beginning to the end of each period. Renin release was measured on 129
occasions in the twelve animals. This represented fifty-nine simultaneous measurements of
renin release from both kidneys and eleven measurements from one kidney only. Four techniques were tested in an attempt to produce renal ischaemic changes.
1. In four animals the main renal artery of one kidney was totally occluded for I hr during
the third and fourth experimental periods. Following release of the clamp in two of these
animals, additional blood samples were taken at the times indicated in Fig. 7.
TABLE
1. The effect on the measurementof standard dog renin (Tubes 7,8 and 9) of addition
of renal lymph (Tubes 4,5 and a); there is no evidence that addition of lymph increased or
decreasedthe apparent renin content beyond that attributableto the renin present in lymph
Standard dog
Tube Lymph (ml) renin (ml)
0.1
0.1
0-1
0.1
0.1
0.1
-
0.1
0.1
0.1
0.1
0.1
0.1
Substrate
(ml)
Buffer
(ml)
0.9
0.9
0-9
0.8
0.8
0.8
0.9
0-9
0.9
Renin units found
0.072
0.035
0.036
2. In three animals a solution of E. coli endotoxin (Difco) in 0.9% NaCl was infused at a dose
of 3-5 mg/kg directly into one renal artery for 10-13 min at the beginning of the third experimental period.
3. In four animals a solution of haemoglobin (0.2-0.3 g haemoglobin/kg of body weight,
prepared in the manner described by Goldberg 1962) was infused into one renal artery during
the third experimental period.
4. In one animal noradrenaline was infused into one renal artery at a rate of 0.1 pg kg-l
min-' for 1 hr during the third and fourth periods.
At the end of the experiment, the animals were killed with intravenous barbiturate; the
kidneys were removed immediately and a transverse slice approximately 5 mm in thickness was
taken from the equator of each kidney and fixed sequentially in 10% formol-saline and corrosive formalin. The interval between the onset of ischaemia and the fixation of the kidneys
varied from 2+ to 4 hr.
RESULTS
Precision of techniques
Renin release. The rate of r e i n release into the renal circulation was estimated from the
product of renal plasma flow and the V-A renin concentration difference (see Methods). Only
162
K . F. Hosie et al.
minor inaccuracy is likely to be contributed by estimation of renal plasma flow (Fig. 1).
Measurement of V-A renin difference is a greater potential source of error. As replicate measurements of plasma renin concentration vary less when extracted and estimated in the same
batch, all renin samples from single experiments (up to twenty-seven in number) were measured
in this way. Twenty-nine replicate estimates of plasma renin concentration varied within
batches with a standard deviation of f8-65%. This value may be used to derive the smallest
difference (0)
which can be detected between two samples: D = tS J2, where S is the standard
deviation of replicate estimates for the technique under consideration and ' t ' determines the
level of significance. For a 0.05 level of significance the smallest difference of renin concentration which is detectable in a pair of plasma samples is 25% and thus single estimates of renin
release based on a V-A difference of less than 25% are likely to be unreliable. 41% of all
estimates made in this study were in this imprecise range. Most of theseoccurred duringcontrol
periods. Large V-A differences of up to 2000% of arterial plasma renin concentration were
usually found during experimental periods (Figs. 3, 7 and 8).
As a test of the quantitative characteristics of the assay technique, serial dilution of two
plasma extracts produced a decrement in velocity comparable with that produced by serial
dilution of standard dog renin (Fig. 2).
Lymph renin assay. Tests of the lymph assay (see Methods) showed:
I . That there was insufficient renin substrate present in lymph to interfere with the assay of
renin. The velocity of angiotensin production on incubation of triplicate lymph samples without added substrate was less than 0.25 ng ml- hr- '. This value represents the limit of sensitivity
for bioassay. Velocities of 1.8, 1.6 and 1.6 ng ml-' hr-' were obtained when substrate added
in standard concentration was incubated with the same volume of this specimen of lymph.
2. A small amount of angiotensinase was present in one lymph sample: 50-70% of added
angiotensin being recovered after incubation for 24 hr. This did not affect the linearity of
angiotensin production measurably nor did it increase or decrease the rate of angiotensin
production when renin was incubated with its substrate (Table 1). Similarly, the quantitative
characteristics of the assay technique were unimpaired (Fig. 2).
3. Lymph did not produce any net inhibitor or accelerator effects on the renin-substrate
reaction, in that addition of lymph to an incubation mixture of dog-renin and substrate did not
increase or decrease the expected velocity of angiotensin formation (Table 1).
4. The quantitative nature of the lymph assay was confirmed by demonstrating that dilution
of lymph produced a comparable decrease in velocity of angiotensin formation to that resulting from the same dilution of standard dog renin (Fig. 2).
'
Stability of anaesthetized dog preparation
No animal developed anuria after the surgical procedure which involved occlusion of the
renal artery and cannulation of the renal vein. Urine volume during the first experimental
period varied between 0-03 and 1.3 ml kidney-' min-' (mean 0.4 ml kidney-' min-I). The
osmolality of urine at this time ranged from 194 to 931 mOsm/kg (mean 478 mOsm/kg) and
the average renal plasma flow for a single kidney during the first experimental period was 3.5
(SD 1.54) ml kg-I min-'. Blood pressure was well maintained except in animals injected with
endotoxin or haemoglobin solution. Mean arterial pressure during the first period of observation was 127 (SD 23) mmHg. With the exception of dogs showing a large change in renin
release (Figs. 7 and 8) the renin concentration in arterial blood remained relatively constant
Renin release
163
throughout the period of observation; the mean renin values for the first and last experimental
period were 35.2 (SD 17.7) and 34-5 (SD 15.2) units/litre respectively.
Microscopic examination of the kidneys
The only clear-cut histological effects were produced by injection of haemoglobin solution
when particulate matter was found in Bowman's capsule together with epithelial atrophic
changes and cast formation in the loop of Henle and in the distal convoluted tubule. These
changes were more marked in the injected kidney. There was no difference in the appearance of
the macula densa or the juxtaglomerular apparatus in control and experimental kidneys or any
experiment.
Studies of plasma renin concentration
Renin was present in each of the 199 samples of plasma examined in a concentration which
varied between 9.4 and 620 units/litre. The sensitivity limit for the renin method with the batch
of substrate used in these experiments is 1.3 units/litre for a 10 ml sample of plasma.
Relation between renal venous and arterial renin
Arterial and renal venous plasma renin values were significantly related (Fig. 3, r = +0.24,
P<0.02) and the relation became closer ( r = +0-498, P<O.Ool) on excluding from the analysis samples which had been taken at a time when renal plasma flow was reduced experimentally
to 30 ml min-' kidney-' or less (Fig. 3). As will be described, reduction of renal plasma flow
may lead to a large increase of renal venous renin concentration (RVR) without measurable
change either in renin release or in arterial plasma renin concentration. Generally, the concentration of renin in renal venous plasma was higher than that in arterial plasma (Fig. 3). For
the 103 observations in which renal blood flow had not been reduced to 30 ml/min or less,
arterial plasma renin concentration was on average 36.5 (SD 18.1, SEM 1-78)units/litre, while
RVR was44*25(SD34.5, SEM 3.4) units/litre ( t = 2.10, P<0.05). Thus, in these circumstances,
83% of the renin present in renal venous blood can be accounted for by renin which has entered
the kidney in arterial blood, although this does not necessarily imply that all renin entering
the kidney in arterial blood passes directly into the renal vein.
On nineteen occasions a significant negative V-A renin difference was present (Figs. 3 and 4).
That is, more renin entered the kidney in arterial blood than left in the renal vein. In two
experiments (Figs. 7 and 8) the V-A difference became negative in the control kidney after
release of the renal artery clamp.
Renin release and renal venous plasma renin concentration ( R V R )
For the group of 129 observations, RVR was also positively related to the rate at which
renin was released into renal blood (r = +0-37, P < O - O l , Fig. 4); although on some occasions
(period 5, Fig. 8), RVR may fall during a rise of renin release.
Relation between renal plasma flow, and R V R
Renal plasma flow was inversely related to RVR (n = 129, r = -0.39, P<O.OOl) and also
to the V-A renin difference ( r = -0.28, P<O-O1). The inverse relation is well shown by the
opposite changes of renal plasma flow and RVR which follow clamping and unclamping the
renal artery (Figs. 7 and 8).
164
K . F. Hosie et al.
10
I
I
I
I
40
100
400
1000
Renal venous plasma renin concentratlon (unlts/l)
F k . 3. Relationship between renin concentration in arterial and renal venous plasma samples
taken at the same time. The points marked with a cross represent measurements taken when renal
plasma flow was 30mlmin-' kidney-' or less. Those marked with a dot were taken when renal
plasma flow was greater than this. Points lying above a 45' line represent a negative V-A renin
difference, those below the line a positive V-A difference.
X
X
X
X
X
X
-I
?(
X
X
x
X
X
x
X
-3'
10
1
I
I
40
100
400
Renal venous plasma renin concentratlon (units/ I )
FIG.4. Relationship between renin release and plasma renin concentration. Points marked with a
cross are based on V-Arenin concentration differences greater than 25%. In those marked with a
dot the V-A difference was less than this.
Renin release
165
Three determinants of RVR
As might be expected from the Fick principle, RVR may increase for three reasons:
(a) because renal plasma flow is reduced (Figs. 7 and 8), (b) because renin release is increased
(Fig. 4), or (c) because arterial plasma renin concentration is increased (Fig. 3). These effects
may be independent: RVR may actually fall during an increase of renin release, as after
removing the arterial clamp (Fig. 8) and, on other occasions (e.g. during arterial clamping in
Fig. 7), RVR may rise during a reduction of renal plasma flow when neither renin release nor
arterial plasma renin concentration change measurably. Reduction of renal plasma flow might
increase RVR partly because renin is released into a smaller minute-volume of blood, and
partly because renin release is itself stimulated by a reduction of renal plasma flow. The second
mechanism seemed relatively unimportant in the circumstances of our experiments, as renin
release was insignificantly related to renal plasma flow (n = 129, r = + O M , P>0.05). This
does not exclude the possibility that renin release is stimulated by a reduction of renal plasma
flow, but it does suggest that the effect does not contribute in major degree to the significant
inverse relation we have found between renal plasma flow and RVR.
-0.04
0
+0,04
i-0.08
Renin release [units hg-'min-')
+0.12
FIG. 5. Relationship between arterial plasma renin concentration and the combined renin release
rates for the two kidneys.
Renin release and peripheral plasma renin concentration
As would be expected if the renal vein was an important route of renin release, the sum of the
release rates for the two kidneys was positively related to the concentration of renin in peripheral arterial plasma (Fig. 5, r = +0.58, P<O.OOl). However, this relationship was only
significant if renal artery clamping experiments were excluded. In these experiments, rapid
changes of renin release preceded changes in plasma renin concentration. For example, removal of the clamp on the renal artery in one animal (Fig. 7) was followed within 1 min by the
highest rate of renin release so far observed (21.8 units/min) and yet peripheral arterial renin
concentration at this time had not increased (19 units/litre). Two minutes later this state of
166
K. F. Hosie et al.
affairs had reversed ; renin release had then decreased and peripheral arterial renin concentration had increased from 19 to 60 units/litre. This was the largest increase of arterial renin
seen.
Renin and blood pressure
Plasma renin concentration was not related to the level of arterial pressure during the control
period (r = +0.215, P>0.05),although a rise of blood pressure occurred after clamping the
renal artery in the two animals whose renin release and arterial renin increased most (Figs. 7
and 8).
Renin in renal lymph
Renin was present in each oftheeighteen samples ofrenallymphexamined (Table2). In all cases
the renin concentration of lymph exceeded that in renal venous plasma taken at the mid-point
ofthe lymph collection (Fig. 6).Tt is ofinterest that the renin concentration ofrenalvenous plasma
Renal
vein
Lymph
FIG.6. Renin concentration of renal venous plasma and renal lymph. The lines connect values
obtained from simultaneous samples.
during a period of renal artery clamping (620 units/litre) was close to that found in lymph
following removal of the clamp (640 units/litre). This suggests that plasma and lymphatic
renin may equilibrate during a period in which the circulation of the kidney is arrested.
Renin was released into single lymphatics at a rate which varied between 0.0088and 0-055
units/min (Table 2). These values were compared with the rate at which renin was released
into renal blood on ten occasions (Table 2). In six of these the V-A renin difference was
Renin release
167
negative but within a range in which measurement was not precise. With this proviso it would
appear that in some circumstances the renal lymphatics may be the only route by which the
kidney makes a positive contribution to the renin present in blood. During the sixth collection
period in Dog 414 (Table 2) the rate at which renin was released into single lymphatics from
the two kidneys (0.068 units/min) was greater than the rate at which it was removed by the
kidneys from blood (0.04units/min), although, here again, the second measurement was not
based on a significant V-A renin difference.
TABLE
2. The concentrationof renin in renal lymph and the rate of release of renin into single lymphatics
as compared with renin release into the renal vein; lymph collections were for periods of 30 min
Lymph
Dog
Sample no.
414
L1
L2
L3
L5
L6
R1
R3
R4
412
R5
R6
R1
R2
R5
41 1
R6
R1
R2
R5
R6
Renin concentration
(units/litre)
190
150
130
240
320
440
300
330
300
300
210
130
640
320
540
640
880
1040
Renin release
Renin release into renal vein
(units kidney-' min- I )
(units lymphatic- min- I )
0.020
0.015
0.013
0.024
0.038
0.043
0.026
0.033
0.025
0.030
0.01 2
0.0088
0.023
0.028
0.032
0.030
0.055
0.051
- 0.47
+0.10
-
+0.14
-
+
0.04
-0.1
-0.18
-0.28
-0.48
+ 5.6
-0.047
Generally, however, the average rate at which renin was released from single kidneys into
blood (0.6 unit kidney-' min-') was considerably greater than the average rate at which
renin was released into single lymphatics (0.028 unit lymphatic-' min-'). Even if allowance is
made for the fact that only one of the five or six hilar lymphatics from each kidney was cannulated, it would still seem that the renal vein is usually the major route by which renin leaves the
kidney.
Eflect of reducing renal bloodJlow
Four methods of reducing renal blood flow were tested (see Methods).
1. During occlusion of the renal artery for 1 hr renal blood flow decreased markedly but did
not cease, RVR increased in each of the three dogs, renin release increased in one of three
dogs, the flow of lymph ceased in both of two, and anuria developed in all of three. Removal of
168
K. I;. Hosie et al.
the clamp was followed by a short period of increased blood flow (Figs. 7 and 8). Renin release
increased markedly after removing the clamp in the three animals. Renal lymph renin concentration also increased during this period in the two animals in which measurements were
made.
FIG.7. The effect on (a) renal plasma flow, (b) renin concentration in renal venous and arterial
plasma, (c) the rate of renin release, and (d) mean arterial, pressure of clamping the left renal artery
for 1 hr in an anaesthetized dog. Arterial renin values are illustrated twice. Points marked with an
additional ring represent significant differences of V-A renin concentration (see text).
2. Following intra-arterial injection of E. coli endotoxin, blood pressure fell within 2 hr to
35,48 and 50 mmHg respectively in the three injected animals. Renal blood flow decreased in
parallel in two of the three animals during this period, and in both reduction of flow was
greater in the injected kidney. Renin release increased slightly in one of these, and remained
unchanged in the other. In the third dog, injection of endotoxin was followed by a small
increase of renal plasma flow and by little or no change in renin release.
169
Renin release
3. Following intra-arterial injection of haemoglobin solution, blood pressure decreased in
the four animals injected. Renal blood flow was also reduced in parallel with the change of
blood pressure. In only one animal did haemoglobin reduce blood flow to a greater extent on
the side injected. There were no distinct changes in renal venous renin concentration or in
Clamp right renul urtery
'
100
20
.-.
Right
.-•
Hours
FIG.8. The effect on (a) renal plasma flow, (b) renin concentration in renal venous and arterial
plasma, (c) the rate of renin release, and (d) mean arterial pressure of clamping the right renal
artery for 1 hr in an anaesthetizeddog. Points marked with an additional ring represent significant
differences of V-A renin concentration (see text).
renin release from either kidney in the four animals. Urine volume was reduced in three of
the four animals and anuria developed in one. Histological examination showed distinct
abnormalities which were most marked in the distal nephron of the injected kidney.
4. In one animal infusion of noradrenaline at a rate of 0.1 pg kg-' min-' for 1 hr into the
renal artery was followed by a reduction in renal plasma flow from 82 to 17 ml/min, by an
170
K . F. Hosie et al.
increase in blood pressure from 112 to 135 mmHg, by an increase of RVR on the infused side
from 23 to 43 units/litre, by a decrease of renin release from 0.64 to 0.32 units kidney-' min-'
on the infused side, and from 0.45 to 0.26 units kidney-' min-' on the control side. Following
the infusion, renin release increased to 0.55 units kidney-' min-' on the infused side, and to
0.33 units kidney-' min-' on the control side.
D I S C U SSI O N
The main interest of these experiments lies in the technical problem of measuring renin release,
in the occasional finding of a higher concentration of renin in arterial than in renal venous
plasma and in the relation between renin concentration of renal lymph and renal venous plasma.
No information has been obtained on the release of renin during the production of ischaemic
changes within the kidney as the expected histological changes did not occur with three of the
four techniques tested. The period of ischaemia or the interval between ischaemia and removal
of the kidney may have been too short. Alternatively, heparin was used as an anticoagulant
and recent work suggests that it may protect the kidney from ischaemic change (see KincaidSmith, Saker & Fairley, 1968).
Limitations imposed by the precision of renin assay
Measurement of renin release largely depends for its accuracy on the precision with which a
small V-A renin difference can be determined. This can be assessed from the variation of
replicate estimates of plasma renin concentration which in our own technique has a SD of
8.65%. Using this value in the manner described in the section on results the smallest V-A
difference which can be determined accurately is 25% (expressed as (V-A/A) x 100). Although
V-A differences larger than this were often found during experimental periods most estimates of
renin release during control periods were based on a V-A renin difference which was less than
25% (Figs. 3, 4, 7 and 8), and it is not, therefore, generally possible to measure in a single
animal the small changes of renin release that occur during control circumstances. However,
smaller changes of renin release could be determined by replicate measurements of renin in
single plasma samples or by measurement of renin release in several animals.
Other methods of measuring renin release are limited in a similar way. In those techniques
where replicate estimates of renin are reported the SD of replicate variation is quoted as 10.7%
(Kaneko et al., 1967) and 14.4% (Vander & Miller, 1964).
Renin activity and renin concentration in the determination of renin release
The calculation of renin release is based on absolute and not relative values of plasma renin
concentration (see Methods). For a given plasma flow, venous and arterial plasma renin
values of 100 and 90 units/litre respectively will produce the same rate of renin release as
venous and arterial values of 20 and 10 units/litre. For this reason it is essential that the units of
renin measured are strictly related to the concentration of renin present. We have confirmed
this point for our own method (Fig. 2).
Much earlier work on renin release has employed the renin activity technique (Skinner,
McCubbin & Page, 1963; Vander & Miller, 1964; Wathen et al., 1965). Changes of renin
activity are not necessarily parallel with changes of plasma renin concentration (see Brown
et al., 1966). The renin activity technique may then give no quantitative information on renin
Renin release
171
release unless standardized against renin concentration. Wathen et al. (1965) and Kaneko
et al. (1967) have calibrated renin activity in this way and express their results in terms similar
to our own.
Renin activity measurements may, however, provide information on the direction rather
than the magnitude of renin release in circumstances in which arterial renin activity and renal
plasma flow do not change markedly (e.g. Skinner et al., 1964). However, difficulty of interpretation will arise when arterial renin or renal plasma flow change. For example, Bunag, Page &
McCubbin (1966a) showed that infusion of noradrenaline into the renal artery increased renal
venous plasma renin activity and also reduced renal plasma flow to less than 50% of its control
value. Their interpretation that renin release had increased would be valid only if the increase
in renin activity was greater than could be accounted for by a two-fold increase in the V-A
plasma renin concentration difference. This point was not established.
The importance of measuring arterial plasma renin in the estimation of renin release
As in earlier studies of renin release (see Introduction), we have shown that on balance a
large proportion of the renin leaving the kidney in the renal vein can be accounted for by renin
which enters the kidney in arterial plasma (Fig. 3). Arterial plasma renin concentration or
activity cannot therefore be ignored in computing either the magnitude or the direction of a
change of renin release. Vander & Miller (1964) have stressed this as a theoretical requirement in measuring renin release. We do not, however, accept their point, that because arterial and
renal venous renin activities are parallel, measurement of venous activity alone is for practical
purposes an adequate indicator of renin release. It is because these values often change in parallel,
that both must be used to compute renin release. A similar objection applies to the technique used
by Bunag, Page & McCubbin (1966b) where changes of renin release are based on changes of
renal venous renin activity. Although RVR and renin release are positively related in a large
series of observations (Fig. 4) the two values may not be equated. An increase of RVR could
as well be due to an increased arterial renin as to an increased renin release. In fact, RVR
and renin release may change in opposite directions in some circumstances (Fig. 8).
Importance of measuring renal plasma pow
A significant inverse relation between renal plasma flow and renal venous renin activity or
concentration has been shown, both in our own and in an earlier study (Kaneko et al., 1968).
Reduction of renal plasma flow may raise RVR without necessarily increasing renin release
(Fig. 7 and Wathen et al., 1965). This point is relevant to the demonstration of increased
renin or pressor activity in the venous blood of ischaemic or diseased kidneys in some hypertensive patients (Judson & Helmer, 1964; Goorno & Kaplan, 1965; McPhaul et al., 1966;
Meyer et al., 1966; Fitz, 1967; Winer et al., 1967; Woods & Michelakis, 1968; Grollman &
Ebihara, 1968). Renal plasma flow was not measured in the majority of these studies and, as
has been stressed (Brown et al., 1965; Fitz, 1967), it is not certain whether the increase in renal
vein activity is due to reduction of renal plasma flow or to an increased release of renin.
Positive and negative V-A renin diference
As in previous studies using the renin activity technique (Skinner et al., 1964; Vander &
Miller, 1964; Nash et al., 1968), the present observations show that renin concentration of
renal venous plasma is generally higher than that in arterial plasma. Thus, as was originally
B
1 72
K, F. Hosie et al.
proposed by Tigerstedt & Bergman in 1898, renin is probably released from the kidneys into
blood. This is further borne out by the positive relation between the rate of renin release and
arterial plasma renin concentration (Fig. 6).
However, on nineteen occasions significantly more renin was present in arterial than in renal
venous plasma. We cannot exclude the possibility that this results from a different recovery of
endogenous renin from arterial and renal venous plasma during extraction, although experiments based on the renin activity method in which the plasma sample is not subjected to
extraction, also suggests that the renin content of arterial or peripheral venous plasma may
occasionally be greater than that of renal venous plasma (see Meyer et al., 1966; Vander &
Luciano, 1967; Kaneko et al., 1967; Winer et al., 1967). Dilution of renal venous renin by
mixture with adrenal, ovarian or testicular venous blood is also unlikely, as, in our experiments,
the cannulae were passed into the renal vein beyond the entry of these vessels.
A more likely explanation of the negative V-A difference is that renin release is a balance
between two processes, removal of renin from blood during its passage through the kidney,
and liberation of renin into blood from the juxtaglomerular apparatus. Generally, the second
process is dominant with the kidney making a positive contribution to renin in blood. Less
commonly, removal of renin may predominate and then the V-A renin difference would be
negative. It is also possible that the liberation of renin from the juxtaglomerular apparatus
may be intermittent, and that for short periods when renin is not being liberated venous renin
concentration may fall below arterial renin, and yet, on average over a prolonged period, the
kidney would release more renin than it removed. The experiments described here give no
information on this point.
Several observations support the idea that the kidney removes renin from blood: a renin-like
enzyme is normally present in urine (Brown et al., 1964a; Anichini & Gross, 1965) and that it is
at least partly derived from arterial renin, is strongly suggested by the appearance of renin in
urine following its injection into a peripheral vein (Houssay, Braun-Menendez & Dexter,
1942; Rapelli & Peart, 1968). Secondly, as was also shown by Houssay and his colleagues,
removal of both kidneys prolongs the survival of renin in blood.
The greatest negative V-A renin difference was seen after a rapid increase of arterial plasma
renin concentration, which reversed a previously positive V-A renin difference in the control
kidney (Figs. 7 and 8). This effect is unlikely to have resulted from delayed mixing of blood
within the kidney, as it persisted in one animal for more than 1 hr. An increase of arterial renin
might reverse the V-A renin difference, either because more renin was removed from bloodas might occur if renin was cleared by glomerular or tubular effects-or because less renin was
liberated from the juxtaglomerular apparatus. The latter is an interesting possibility, which is
suggested by experiments of Vander & Geelhoed (1965) which showed that infusion of angiotensin was capable of reducing renal vein renin activity. As the authors suggest, a negative
feedback system may exist in which release of renin from the kidney may, via angiotensin,
inhibit further renin release.
Renal lymph
It is important to distinguish the concentration of renin in renal lymph from the amount of
renin leaving the kidney by the lymphatic route. Although the concentration of renin in lymph
is high, the flow of lymph is slow, and thus only small amounts of the enzyme may leave the
kidney by this route (Lever & Peart, 1962). As in these earlier experiments, the concentration of
Renin release
173
renin in renal lymph was found to be higher than that in renal venous plasma. This suggests
either that renal venous renin is derived from lymphatic renin by simple diffusion, or that
both are derived from a common source, such as the juxtaglomerular apparatus, where renin
concentration is higher still. Our observations do not suggest that lymphatic renin is derived
from plasma renin unless active removal of water or secretion of renin from blood is involved,
and these effects do not seem to occur with other proteins which pass from plasma to renal
lymph (Mayerson, 1963).
The relative amount of renin leaving the kidney by the lymphatic and renal venous routes
has not been established as it was not possible to collect all lymph draining from the kidney.
Generally, however, our results suggest that more renin leaves in renal venous plasma than in
lymph, although, during periods in which the V-A renin difference is negative, the lymphatic
route would be the only one by which renin could be added to the circulation. The observations
of Skinner et al. (1963) suggest that in other circumstances the angiotensin content of renal
venous plasma may be greater than that in thoracic duct lymph.
ACKNOWLEDGMENTS
We thank Mrs Eleanor Prentice, Miss Anne Robinson and Mrs M. Thurgo for their help.
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