Direct Radioimmunoassay of Human Renin
Comparison with Renin Activity in Plasma and Amniotic Fluid
TAM T. GUYENE, M.D.,
FRANCOIS X. GALEN, PH.D., CLAUDINE EJEVAUX, PH.D.,
PIERRE CORVOL, M.D., AND JOEL MENARD, M.D.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
SUMMARY Human plasma and amniotic liquid were actirated by dialysis at pH 33. Then, renin before
and after acidification was determined by two methods: enzymatic actirity measurement, and direct radioimmunoassay. The Identity between nonactivated and actuated renin in plasma and amniotic fluid on the one
hand, and pure renin on the other, was demonstrated by the dilution cones in radioimmunoassay. After
acidification, mean plasma renin activity in 17 patients with high renin activity rose from 26.8 ± 11.7 pinoles A
I ml"1 h 1 to 67.9 ± 29.3 praoles A I ml'1 h~\ whereas the mean renin concentration tested by direct radloimraunoassay remained constant at 13.8 ± 10.5 and 14.8 ± 11.2 fmol/ml before and after acidification respectively. In amniotic fluid, renin activity Increased from 9.7 to 227 pmoles angiotensln I/ml/h, but the renin concentration did not change. Direct radioimmunoassay of renin may therefore be considered as measuring total
renin, regardless of its enzymatic activity. In 12 hypertensive patients undergoing bilateral renal-vein
catheterization, the direct measurement of renin was very significantly correlated to the non-activated
(r = 0.883) and activated renin values (r = 0.963). (Hypertension 2: 465-470, 1980)
KEY WORDS • human renin radioimmunoassay
blnephrectomized patient renin • pepstatin
renin activation
F
IFTEEN years after the initial description
of the methods currently used for measuring
renin (E.C-3-4-99-19) in human plasma 11
and despite their successive improvements,*"5 the investigation of this enzyme is still limited by many
methodological problems. Thus, plasma renin activity
(PRA) is not measured in the presence of a constant
excess of renin substrate. To control this variable element, heterologous substrates extracted from sheep,4
or oxen* are commonly used, but compared to human
angiotensinogen, they introduce differences in enzymes kinetics. The amount of angiotensin I released
from renin substrate through the enzymatic property
of renin in plasma or amniotic fluid can be greatly
changed by several processes generally designated as
"renin activation," i.e., refrigeration,6'7 acidification,8"1' or addition of proteolytic enzymes.14"11 Such
activation is not necessarily linked to conversion of the
forms of higher molecular weight renin in human
plasma12 and kidney2*1 u into lower molecular weight
amniotic fluid
forms, but could be due to a minor structural change
in the renin molecule, or to the destruction of a renin
inhibitor.
The preparation of highly specific human renin antibodies" offers new ways of avoiding these problems.
With these antibodies, we prepared an affinity
chromatography gel in order to separate human
angiotensinogen from human renin in a single step.
We also used antibodies to characterize renin in
plasma from binephrectomized patients. Finally, we
set up a direct renin radioimmunoassay and compared
the number of immunoreactive molecules present in
plasma and amniotic fluid to the enzymatic activity
before and after activation.
Methods
Preparation of Renin Substrate
Renin-free human angiotensinogen was prepared by
affinity chromatography using immobilized antihuman-renin-antiserum. This immunoadsorbant was
obtained by coupling 10 ml of rabbit anti-humanrenin-antiserum brought to pH 8.0 with 100 ml
Sepharose 4 B (Pharmacia) according to March et
al.M After overnight shaking at 4°C, the gel was
washed with 1 M glycine, water, 2 M. acetic acid, 6 M
urea, and 0.1 M phosphate buffer at pH 7.5. The
efficiency of the gel was checked by its ability to bind
From INSERM and UER des Sciences Pharmaceutiques et
Biologiques, Paris, France.
Supported by grants from Institut National de la Sante et de la
Recherche Medicate, Direction Generate de la Recherche Medicale,
and Association Claude Bernard.
Address for reprints: Tam T. Guyene, M.D., INSERM U-36, 17,
me du Fer-a-Moulin, 75005 Paris, France.
465
466
HYPERTENSION
95% radioactive iodinated human renin. Human
plasma was obtained from women taking oral contraceptives; 130 ml of plasma were added to 130 ml of
0.2 M phosphate buffer, pH 7.5. The sample was then
gently shaken for 3 hours at 4°C together with 90 ml
Sepharose-anti-human-renin-antiserum. After 5
minutes of centrifugation, the supernatant was
dialyzed overnight against 0.1 M phosphate citrate
buffer pH 5.7 containing 2% disodium ethylenediamine tetraacetate acid. The 230 ml sample was
then stored at -24°C in 5 ml aliquots.
Renin Activation Procedures
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Activation of renin in amniotic fluid or plasma was
performed by 24 hours of dialysis at 4°C against 0.1
M citrate-phosphate buffer, pH 3.3, followed by 24
hours of dialysis against 0.1 M phosphate buffer, pH
7.4. Control samples were dialyzed for 48 hours at pH
7.4. Since acidification destroys renin substrate, the
angiotensinogen concentration in acidified plasmas
was restored by adding 275 pmoles of human
angiotensinogen (see above). The same amount of
angiotensinogen was used for renin enzyme activity
determination in amniotic fluid.
Radioimmunoassay (RIA)
Plasma Renin Activity
PRA was determined by the amount of angiotensin I
generated at the optimum pH, pH 5.7 and measured
by RIA" with a standard curve ranging from 5 to 80
pg; 250 ^1 plasma or 100 fi\ amniotic fluid were incubated for 30 minutes at 37°C in a total volume of 500
jul. Results are expressed in pmole angiotensin I
generated per milliliter (ml) per hour.
VOL 2, No 4, JULY-AUGUST
Angiotensinogen Determination
An exhaustion method that used cadaver kidneys as
a source of renin1* was used to measure angiotensinogen concentrations, which were expressed in
angiotensin I equivalent generated per milliliter, on
the assumption that 1 mole of angiotensinogen generates 1 mole of angiotensin I.
Direct Renin Radioimmunoassay
Direct renin RIA was performed as described" on
100 MI plasma or 10 fi\ amniotic fluid. Purity of the
iodinated renin was checked by Agarose-Acrylamide
gel filtration (fig. 1), and by isoelectric focusing on
polyacrylamide gel electrophoresis according to Doerr
and Chrambach.*" A single symmetrical peak was obtained with an isoelectric point 6.6. Results were expressed in fmble of enzyme. Variance analysis was
performed to demonstrate the linearity of the standard
curve and of the displacement curves for activated and
nonactivated renin, and the slopes of these curves were
compared.
Inhibition of Plasma Renin by Human RenJn Antibodies
Plasma (250 jil) from binephrectomized patients
were preincubated for 24 hours at 4°C with an excess
of antirenin antiserum (25 nl of a 1/100 dilution) at
pH 5.7. Then PRA was determined at pH 5.7 in a 500
y\ volume, as described above. In control experiments,
preincubation was run in the absence of antiserum. It
was verified that under these conditions the antiserum
concentration used completely inhibited standard
human renal renin (Medical Research Council) and
renin from normal plasma.
cpm x 10 6
per fraction
38 0 0 0 M W
Ovolbumin
Void Volume Albumin
I
4 5-1
1980
•
•
I otChymotfypsinogen
HI
•
3.0
1.5
FIGURE 1. Purification of iodinated renin by
filtration on Agarose-Acrylamide (AcA 44).
Iodinated renin was applied on a 91 X 25 cm
column. Upward elution was developed in 0.1 A/
phosphate buffer pH 7.5. The fractions in the
brackets were pooled and used as a renin tracer.
50
100
FRACTIONS NUMBER
150
467
RADIOIMMUNOASSAY OF HUMAN KENW/Guyene et al.
Results
Preparation of Renio-Free Substrate
Angiotensinogen prepared by affinity chromatography was obtained with 52% recovery whereas
renin was completely eliminated (table 1). No angiotensin I formation could be detected even when incubation lasted for as long as 6 hours. To demonstrate that activated and nonactivated renin were
removed from plasma by this affinity chromatography, plasma eluted from the gel was acidified and
then incubated with exogenous angiotensinogen. The
absence of angiotensin I generation demonstrates that
exposure to immobilized antirenin antibodies removes
both nonactivated and activated renin from plasma. It
also shows that acidification of such plasma is not able
to unmask an angiotensin I-forming enzyme distinct
from renin.
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Direct RIA of Activated and Nonactirated Renin
Figure 2 illustrates the dilution curves, in direct
RIA, for plasma and amniotic liquid before and after
acidification. The slopes of these curves are identical
to the standard curve constructed with pure renin.
This confirms the immunoreactive identity of renin
(activated or nonactivated) in plasma and amniotic
liquid with pure enzyme. Renin inhibitor pepstatin did
not affect the RIA of renin at a 10~5 M concentration.
Thus, the standard curve constructed in the presence
or absence of this pepstatin concentration was indistinguishable.
The influence of acid activation of plasma renin on
the generation of angiotensin I is shown in figure 3.
There was a 253% mean increase in renin enzymatic
activity after acidification of the plasma from 17 high-
TABLB 1.
Plasma
Before
After
affinity
chromatography
Renin-Free Angioteneinogen
Total
Volume angiotensinogen Yield
(ml)
(%)
(nmole)
Total
renin
(pmole
Al/hr)
130
440
100
110
260
226
52
undectatable
renin hypertensive patients. By contrast, the mean
level of the immunoreactive renin tested by direct RIA
did not rise after acidification. The same observation
applied to plasma obtained before surgery from a
patient suffering from a renin-secreting tumor which
allowed purification of human renin." In this subject,
175.3 pmoles of angiotensin I ml 1 h 1 were generated
before acidification and 2153.8 pmoles ml"1 h~l
afterward. There were 1605 fmoles ml"1 of renin found
before and 1456 fmoles after acidification.
In amniotic fluid, a dramatic increase in renin enzymatic activity followed acidification (from 9.7 to
227 pmoles angiotensin I/ml/hr, but immunoreactive
renin remained unchanged (457 and 462 fmoles ml"1
before and after).
Correlation Between Enzymatic Assay and Direct RIA
In figure 4, renin enzymatic assay is plotted against
renin direct RIA in 36 plasmas from 12 patients undergoing bilateral renal-vein catheterization. Renin
secretion was stimulated before catheterization by
sodium depletion (four cases) or captopril treatment
(eight cases). To avoid inadvertent cryoactivation,
100-,
Acidified
—•• Non acidified
o
o
o 50
N
0.56
2.5
1.12
"^Amniotic liquid
Plasma
Pure renin
2.25
4.5 f mol renin
10
20 ul sample
FIGURE 2. Direct radioimmunoassay of human renin in
plasma and amniotic fluid before and after acidification. The
abscissa gives the logarithm scale for standard human renin
(fmol) or sample volumes (ul); the ordinate, \B/Bo\ X 700,
is the percentage of bound radioactivity in the presence and
absence of unlabeled renin. Pure renin (1.12 to 9 fmol is
denoted by connected black circles; amniotic fluid before
acidification by connected black squares, and after acidification (2.5 to 15 ul) by connected open squares. Plasma before
acidification is given by black triangles and afterward by
open triangles (acidification = 1.25 to 7.5 pi). This high
renin plasma was obtained from a patient sufferirig from a
renin tumor before surgery (see text).
HYPERTENSION
468
ENZYMATIC RADIOIMMUNO
ASSAY
ASSAY
E 100 o
E
a
75
VOL 2, No 4, JULY-AUGUST
1980
each plasma sample was immediately divided in
several aliquots for the measurements of activated,
and nonactivated rehin, and for direct renin RIA.
Both activated and nonactivated renin were very
significantly correlated to immunoreactive renin
(/• = 0.883, n = 36; after activation: r = 0.963,
n = 35). Activated and nonactivated renin were also
significantly correlated (r = 0.538).
Inhibition of PRA in Anephrk Patients
25
Z
LU
a.
NA A
NA A
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FIGURE 3. Mean variation in plasma renin before and
after acidification. Renin was measured by two methods: enzymatic assay (pmol angiotensin I generated/ml/hr) and
direct radioimmunoassay (fmol/ml) in plasma from 17 high
renin hypertensive patients before (NA) and after (A)
acidification.
25
50
PLASMA &ENIN [f mol ml" 1 )
FIGURE 4. Correlations between the enzymatic assays and
the radioimmunoassay of plasma renin. Plasma renin
measured by radioimmunoassay was plotted against
Angiotensin I liberated by nonactivated and activated
plasmas.
The lack of sensitivity of the present direct RIA did
not allow measurements of renin in plasma from
binephrectomized patients. The PRA of such three
plasmas was 0.953, 0.123 and 0.253 pmoles angiotensin I/ml/hr. The similarity between renin from normal and binephrectomized patient plasma was
demonstrated by complete inhibition of PRA in such
plasmas preincubated with an excess of antirenin antiserum.
Discussion
The angiotensinogen preparation described here
enables selective removal of renin from plasma in a
rapid reproducible way. The same affinity gel was used
another three times to prepare other batches of reninfree substrate with similar recoveries. Since PRA
measurement methods usually underestimate the
renin concentration in plasma, it is worth working out
a method that provides renin-free angiotensinogen and
is suitable for the measurement of renin in the
presence of a constant excess of homologous substrate. Use of an aminohexyl-pepstatin-sepharose
column" or a concanavalin A-Sepharose column"
was suggested Earlier for this purpose; nevertheless,
although the pepstatin column was very effective in
removing a large amount of renin, affinity chromatography using antirenin antibodies made possible
more complete renin extraction, when a small amount
of endogenous enzyme was present in plasma. This
renin-free angiotensinogen should be used rather than
plasma from binephrectomized subjects, since such
plasma frequently contains a small amount of reninlike enzyme, even in the absence of activation."""
In our study, renin activity found in plasma from
binephrectomized patients was completely inhibited
by specific renin antibodies. It brings another argument in favor of the presence of circulating renin
similar to renal renin in such patients.
For the first time, direct renin RIA allows quantification of the number of immunoreactive molecules
in human plasma. The present results emphasize the
very low renin concentration in plasma (fmole
range/ml). The assay used here is not yet sufficiently
sensitive to detect renin in normal patients, however,
and our studies are currently limited to patients with
high PRA, i.e., more than 20 pmoles angiotensin I
ml"1 h"1 after acidification. The renin activation by
acidification previously reported both in plasma and
amniotic liquid is confirmed here. This activation
process varies from one plasma to another, but the
RADIOIMMUNOASSAY OF HUMAN RENIN/Guyene et al.
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mean percentage increase of renin activity observed in
our high renin subjects is of the same order of
magnitude as the increases observed by other authors,
using several activation methods.87- M
In the absence of activation, PRA measurement is
generally correlated to circulating angiotensin II."
This correlation suggests that the PRA level is a
valuable and practical index to the state of activation
of the renin-angiotensin system. This index is the main
element supporting our present concepts of the influence of posture, sodium diet, and drugs like betablockers on the degree of response of the reninangiotensin system. Nevertheless, measurement of the
number of immunoreactive renin molecules secreted
by normal or pathological kidneys under different
conditions remains necessary for complete investigation of this system. Until the present RIA was worked
out, however, such measurement was impossible.
In this study of 36 venous plasma samples from
renal veins and inferior vena cava, we found a very
significant correlation between enzymatic and immunoreactive renin, in activated plasmas and nonactivated plasmas. In mouse plasma as in human
plasma, Michelakis et al.'B and Mailing and Poulsen40
found a high correlation between nonactivated renin
and immunoreactive renin.
The potential usefulness of direct renin RIA as a
tool for determining true renin secretion by the kidney
is demonstrated by the renin level found in the plasma
of our patient with a renin-secreting tumor. In such
cases, when the amount of renin really present in the
circulation is very high, the inappropriate conditions
for the enzymatic reaction lead to underestimation of
renin in the enzymatic assay of activated and nonactivated plasmas. High renin is not a rare condition in
patients with severe hypertension, and renin-secreting
tumors among such patients would certainly be more
easily detected by the renin RIA. Two hypotheses can
be suggested to explain the fact that acidification increases renin enzymatic activity but leaves its immunoreactivity unchanged:
1. Kinetic changes in the renin-substrate reaction
(by the removal of an inhibitor or the unmasking of an activator).
2. Changes in the renin molecule that passes
from an inactive to an active state without any
change in the total number of immunoreactive
centers.
According to the first hypothesis, there would be no
variation in renin structure during activation but an
increase in the affinity of renin for its substrate. Such a
change should lead to a variation in kinetics during the
enzymatic assay of renin. The direct renin RIA, on the
other hand, would remain constant, whether or not an
inhibitor was present, as demonstrated by the absence
of any change in the renin RIA standard curve in the
presence of the pepstatin concentration (10~8 M) which
inhibits the enzymatic activity of human renin.41 The
hypothesis of a kinetic change caused by inhibitor
removal has been investigated in plasma by Derkx et
469
al.' and Atlas et al.43 and in amniotic fluid by Shulkes
et al.,48 Morris,19 and Ito et al.*°None of these authors
found any significant modification in Km after renin
activation, which rules out the hypothesis of a competitive inhibitor removal. However, these results fit
with the hypothesis of a noncompetitive inhibitor
removal.
Also attractive is the hypothesis of renin activation
due to a change in renin conformation, which would
require the antibody to make no distinction between
the two renin forms. In our present study, the fact that
the dilution curves are identical in renin RIA before
and after acidification argues in favor of this second
hypothesis. Our data are consistent with a possible
change in conformation that does not alter immunoreactivity. Such an assumption is in good agreement with the results of Boyd,12 who only found a
minor change in molecular weight between nonactive
and active renin in plasma. Furthermore Shulkes et
al.,48 Morris,1' and Ito et al.10 reported close similarity
of the physiochemical properties of nonactivated and
activated renin in amniotic fluid. The discrepancy
between immunoreactivity and biological activity is
rather a common feature of polypeptides and was the
origin of the discovery of hormonal precursors.44 In
the renin-angiotensin system proper, a discrepancy
between immunological and enzymatic methods was
pointed out by Egenna et al." They worked out an
angiotensinogen RIA that measures both active angiotensinogen and inactive Des-angiotensin I-angiotensinogen and that under certain circumstances differs from the enzymatic assay.44
Another important application of renin antibodies
is the characterization of angiotensin-forming enzymes found in biological fluids or extrarenal tissues.
Our study clearly shows the close similarity and the
immunological identity between amniotic and plasma
renin. Even when the direct RIA cannot be applied
due to the low amount of angiotensin-forming enzyme, the complete inhibition of enzyme activity by an
excess of antibody is in favor of the immunochemical
similarity of the enzymes, as shown for renin in
plasma from binephrectomized patients.
In conclusion, the new methodology developed
through the use of specific renin antibodies has helped
to throw light on the renin-angiotensin system in
plasma and tissues. More sensitivity in our present
methodology is nevertheless needed to enable investigation of conditions under which renin is secreted
in normal or low amounts.
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470
HYPERTENSION
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Direct radioimmunoassay of human renin: comparison with renin activity in plasma and
amniotic fluid.
T T Guyene, F X Galen, C Devaux, P Corvol and J Menard
Hypertension. 1980;2:465-470
doi: 10.1161/01.HYP.2.4.465
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Copyright © 1980 American Heart Association, Inc. All rights reserved.
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