Evidence for an Extrarenal Source of
Inactive Renin in Rats
YUTAKA Doi,
ROBERTO FRANCO-SAENZ, AND PATRICK J. MULROW
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SUMMARY We studied the source of inactive renin in plasma by investigating the changes of active
and inactive renin after bilateral nephrectomy in the rat. Active renin rapidly decreased after
bilateral nephrectomy, with a half-life of approximately 15 minutes. Inactive renin, on the other hand,
was 20.96 ± 1.63 ng/ml/hr before nephrectomy and gradually increased to reach a peak at 20 hours
after nephrectomy (193 ± 62 ng/ml/hr). The molecular weight of active renin was approximately
40,000 and that of inactive renin was approximately 60,000 on a Sephacryl S-200 column. Inactive
renin was separated from active renin by a Cibacron blue column, and the 0 time inactive renin eluted
in the same fractions as the inactive renin from 20 hours after nephrectomy. The pH optimum of
inactive renin in rat renin substrate was between 5.5 and 7.5, which differs from the optimal value of
pepsin or cathepsin D. The increase of inactive renin in nephrectomized rats was not prevented by
removal of the salivary glands, uterus, spleen, pancreas, stomach, intestines, adrenal glands, or
pituitary. In summary, inactive renin is present in the anephric rat and does not appear to be
converted to active renin in the peripheral blood. The source and control of this extrarenal inactive
renin are still unclear, but this renin is secreted in the rat within hours after nephrectomy.
(Hypertension 6: 627-632, 1984)
KEY WORDS • inactive renin
• prorenin
• nephrectomy
S
INCE Lumbers1 first reported the existence of
inactive renin in amniotic fluid by acid activation, several other investigators have demonstrated the presence of an inactive form of renin in
plasma that can be activated by either acid activation,2"9 cryoactivation,10"12 trypsin,13"'3 pepsin,9 cathepsin D,' 6 plasmin,17"19 or kallikrein.20-21 The source of
plasma inactive renin is still controversial. Although
Takii and Inagami22 isolated inactive renin from hog
kidneys and Atlas et al.23 found inactive renin in the
perfusate of human kidney, others have found inactive
renin in anephric patients.8- 24~26 In humans, active and
inactive renin falls rapidly after nephrectomy.27 The
study of the source of inactive renin has been difficult
because of the lack of a good animal model.
Recently Barrett et al.28 showed trypsin activation of
rat inactive renin. Because of the increased concentration of trypsin inhibitors in rat plasma, a much higher
concentration of trypsin is needed to demonstrate inactive renin in rat plasma. The same group of investigators demonstrated a parallel disappearance of active
• angiotensin
• rat
and inactive renin after nephrectomy in the rat. However, complete occlusion of the renal veins and arteries
resulted in a significant increase of inactive renin.29 In
the present study, we investigated the changes of rat
active and inactive renin after bilateral nephrectomy
and studied some of the characteristics of rat inactive
renin. Also, we studied the effects of anesthesia, sodium loading, and infusion of angiotensin II (ANGII) on
the levels of active and inactive renin after nephrectomy.
Material and Methods
Sprague-Dawley female rates weighing 190 to 220 g
were maintained on regular Purina Chow diet and underwent bilateral nephrectomy under pentobarbital anesthesia (30 mg/kg). To study the effect of anesthesia
on the levels of active and inactive renin, a group of
rats underwent pentobarbital anesthesia without nephrectomy. Twenty hours after pentobarbital anesthesia, active renin was lower in intact rats (5-8 ng/ml/hr)
whereas inactive renin was the same as zero time. The
sodium loading was performed by the addition of 1 %
sodium chloride to the drinking water 5 days before
nephrectomy. The ANG II (20 mg/kg/min) (Sigma
Chemical Company, St. Louis, Missouri) was infused
continuously into the jugular vein by osmotic mini
pump (Alzet 2001) starting 2 days before nephrectomy. Blood samples were obtained immediately after
From the Department of Medicine, Medical College of Ohio,
Toledo, Ohio.
Address for reprints: Roberto Franco-Saenz, M.D., Division of
Endocrinology, Department of Medicine, Medical College of Ohio
at Toledo, Toledo, Ohio 43699.
Received August 29, 1983; revision accepted March 21, 1984.
627
628
HYPERTENSION
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the rats were decapitated at frequent intervals from 0 to
48 hours after nephrectomy. Active renin was measured by radioimmunoassay (RIA) of angiotensin I
(ANG I) by a modification of the method of Haber et
al.30 Fifty fil of plasma was incubated with 5 fxl of 8hydroxyquinoline solution (0.34 M), 5 /xl of dimercaprol (2%), 25 pi of EDTA (4%), 90 /il of Tris acetate
buffer (0.1 M, pH 7.4), and 75 /xl of rat renin substrate. The renin substrate was obtained from rats at 48
hours after nephrectomy. The incubation was carried
out for 1 hour at 37° C at pH 7.4. Aliquots (50 /il) were
used for the RIA of ANG I. Total renin was measured
by the trypsin activation method. Trypsin (SigmaType
III) was prepared immediately before use in Tris acetate buffer (0.1 M, pH 7.4). Plasma was incubated
with trypsin (5 mg/ml plasma) for 1 hour at 4° C. The
reaction was stopped by the addition of lima-bean trypsin inhibitor (Sigma Type II-L) (5 mg/1 ml of plasma).
Inactive renin was calculated as the difference between
total renin and active renin. To make certain that the
trypsin and lima-bean trypsin inhibitor were not interfering in the RIA of ANG I, trypsin plus trypsin inhibitor were first mixed and then incubated with or without
substrate, and an aliquot was tested on the RIA. No
angiotensin was measured. Also, trypsin inhibitor plus
substrate did not generate ANG I. Moreover, increasing the concentrations of substrate had no effect on the
levels of inactive renin.
The molecular weight of renin was estimated by
Sephacryl S-200 column (Pharmacia) 1.5 x 99 cm
with the use of bovine serum albumin, ovalbumin,
chymotrypsin A, andribonucleaseA as the molecular
weight standards (Pharmacia). Rat plasma (1 ml) was
placed on the column and eluted with 0.05 M Tris
chloride buffer pH 7.4 containing 0.1 M sodium chloride and 0.02% sodium azide. Then 1 ml fractions
were collected, and in each fraction the concentration
of trypsin inhibitor was measured by using N a-benzoyl-L-arginine-p-nitroanilide as a substrate of trypsin
and by using a colorimetric assay.31 Then 200 /xl of
each fraction was activated with different doses of
VOL 6, No 5, SEPTEMBER-OCTOBER 1984
trypsin (0.1 to 5 mg/ml) according to the concentration
of trypsin inhibitor present in each fraction.
Cibacron blue F 3G-A affinity column size (1.5 x
25 cm) (Pharmacia Sepharose Blue CL-6B) was used
for separation of active and inactive renin according to
the method of Yokosowa et al.32 The column was eluted with 0.02 M sodium phosphate buffer pH 7.1. The
concentration of sodium chloride was changed in stepwise increments from 0.2 to 1.4 M. Inactive renin in
the samples was activated by the addition of trypsin,
2.5 mg/ml of sample, and the samples were incubated
as before. The recovery of active renin was approximately 90%, whereas the recovery of inactive renin
was 50%. The results of the experiments are expressed
as the means ± the standard error (SEM). Statistical
analysis was performed by Student's t test. Significance was defined as a p value less than 0.05.
Results
The time course of active and inactive renin after
bilateral nephrectomy is shown in Figures 1 and 2. At 0
time, active renin was 13.52 ± 2.3 ng/ml/hr, and
inactive renin was 20.96 ± 1.64 ng/ml/hr. Active
renin decreased rapidly after bilateral nephrectomy,
with a half-life of approximately 15 minutes. At 16
hours after nephrectomy, active renin was close to
zero. On the other hand, inactive renin gradually increased after nephrectomy and reached a peak at 20
hours (193 ± 62 ng/ml/hr). It then slowly decreased.
At 48 hours after nephrectomy, inactive renin was still
significantly higher than at 0 time (33.69 ± 1.31 ng/
ml/hr vs 20.96 ± 1.63 ng/ml/hr).
The molecular weight of active and inactive renin
before and after nephrectomy is shown in Figures 3
and 4. The molecular weight of active renin was approximately 40,000 and that of inactive renin was approximately 60,000 at 0 time. However, at 20 hours
after nephrectomy (Figure 4), the peak of active renin
completely disappeared, and an extremely high peak
of inactive renin was apparent. The molecular weight
:
200-
/ \
e 15-
*P<005
**P<001
N-4
150-
z
o
* P<0 05
* * P<0 001
< "H
100-
z
o
z
z
|
\
\
50 -
\
Ns/
0 2
16
20
-ND-
-ND-
24
36
•II— N D -
TIME (HOURS)
FIGURE 1. Time course of active renin after bilateral nephrectomy in female rats. ND = undetectable.
16
20
24
TIME (HOURS)
FIGURE 2. Time course of inactive renin after bilateral nephrectomy in female rats. The asterisk indicates a significant
difference from 0 time inactive renin.
629
EXTRARENAL INACTIVE RENIN/Do; et al.
8ACTIVE RENIN
7-
I
a
9
110
5
120
FRACTION NUMBER
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FIGURE 3. Molecular weight of active and inactive renin in 0
time plasma calculated from their elution volume on a Sephacryl S-200 column. Inactive renin was located by trypsin activation of the eluates from the column. The trypsin concentration
used was adjustedfor the amount of trypsin inhibitor present in
each fraction. Fractions were collected in 1 ml aliquots.
LU
O
4H
o
O 3UJ
2-
1-
of inactive renin 20 hours after nephrectomy was approximately 60,000.
To further characterize and purify inactive renin, we
used a Cibacron blue affinity column. Figure 5 shows
the elution pattern of active and inactive renin at 0 time
on a Cibacron blue affinity column. Active renin eluted
in the first peak between Fractions 12/25, and inactive
renin eluted in the last peak between Fractions 92/105.
Figure 6 shows the elution pattern of inactive renin 20
hours after nephrectomy. There was no active renin
peak, and the inactive renin peak eluted in the same
fractions as that of the zero time inactive renin.
70
80
90
100
FIGURE 4. Elution profile of active and inactive renin on a
Sephacryl S-200 column from plasma collected 20 hours after
nephrectomy. Trypsin activation was done as in Figure 3.
Figure 7 shows the pH optimum of active (0 time)
and inactive renin (at 20 hours after nephrectomy) following sequential chromatography on Sephacryl S-200
and Cibacron blue affinity columns. The pH optimum
of inactive renin was from 5.5 to 7.5, the same as that
1.4M Nad
PROTEIN
ACTIVE
RENN
2-
INACTIVE
RENJN
F
<r
W
o
o
o
10.5-
UJ
20
30
40
110
FRACTION NUMBER
50
60
70
FRACTION NUMBER
80
90
100
110
120
FIGURE 5. Elution profile of active and inactive renin ofO time plasma chromatographed on
Cibacron Blue affinity column. Protein concentration in each fraction was measured by
spectophotometry, and inactive renin was measured by trypsin activation of the eluates.
HYPERTENSION
630
VOL 6, No 5, SEPTEMBER-OCTOBER 1984
i.4MNacl
PROTEIN
ACTIVE
RENIN
INACTIVE
RENIN
10
20
30
40
50 60
FRACTION NUMBER
70
90
100
110
120
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FIGURE 6. Elution profile of active and inactive renin of plasma collected 20 hours after
nephrectomy on a Cibacron Blue affinity column. Inactive renin was measured by trypsin
activation in the same manner as in Figure 5.
ACTIVE
REMN
100
MACTIVE
RENIN
o
50-
3.3
4.5
55
6.5
7.5
8 5
95
INCUBATION pH
FIGURE 7. Optimum pH of active renin from 0 time plasma
and inactive renin from plasma obtained 20 hours after nephrectomy. Renin concentration is expressed as % of maximum
of angiotensin I generated during the incubation. The active
and inactive renins were first purified by sequential chromatography on Sephacryl S-200 and Cibacron Blue affinity columns.
of active renin. To investigate the possible source of
inactive renin, various organs were removed at the
time of nephrectomy. Figure 8 shows that removal of
the salivary glands, uterus, adrenals, and pituitary at
the time of nephrectomy did not prevent the increase in
inactive renin 20 hours later. Similarly, removal of the
pancreas, stomach, and spleen in one rat and removal
of the intestines in another did not blunt the rise in
inactive renin 20 hours later.
To determine if inactive renin levels are influenced
by physiological mechanisms that inhibit the secretion
of active renin, we examined the effect of sodium
loading and ANG II on inactive renin after nephrectomy. Figure 9 shows that 5 days of sodium loading
and ANG II infusion could not prevent the increase of
inactive renin in nephrectomized plasma.
O-Tme
O-TVne
200
20 hours after nephrectomy
CD Active rerun
17771 kractrve rerun
20 hews alter nephrectomy
NS
~ 200
I—i active renin
] nacttve renn
NS
i
100
1 10 °
N=3
N=3
Kidney
Orty
I
Safvary
Gland
uterus
Adrenal
Control
Nephredomy
Nephrectomy Nephrectomy
Pituitary
Removal of Organs -
FIGURE 8. Effect of organ removal on the concentration of
active and inactive renin 20 hours after nephrectomy. The organs were removed at the same time as nephrectomy.
(1% water)
(20no/Vg/mtn)
FIGURE 9. Effect of sodium loading for 5 days before nephrectomy on the levels of active and inactive renin, and the
effect angiotensin II infusion begun 2 days before and continued
until the rats were sacrificed (n = 3).
EXTRARENAL INACTIVE RENIN/Doi et al.
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Discussion
The presence of inactive renin in human plasma
after bilateral nephrectomy has been reported by several investigators. 824 " 26 In humans, active and inactive
renin decrease rapidly after nephrectomy, but the halflife of inactive renin is significantly longer than that of
active renin.27 In the rat, Barrett et al.29 reported a
parallel decrease in active and inactive renin after bilateral nephrectomy. However, their results are difficult to interpret since, in contrast to normal rats in
which inactive renin accounts for approximately 80%
of the total renin, their nephrectomy experiments
showed that inactive renin was lower than active renin
and accounted for only 33% of the total renin. It is
possible that differences in the experimental design
may account for the discrepancy in the results, since
they reported that stress caused significant stimulation
of active renin with a reciprocal decrease in inactive
renin. It is conceivable that stress may have influenced
the levels of active renin in their nephrectomy rats, and
therefore the experimental conditions were different
than ours. Furthermore, they measured the levels of
active and inactive renin during the first hour after
nephrectomy and 24 hours later, whereas in our experiments we followed the levels at frequent intervals for
48 hours.
There is considerable variation in the literature regarding the normal levels of inactive renin in the
rat. 28 ' w ' 33 This variability is probably the result of different experimental conditions as well as differences in
the methods employed to activate inactive renin,
which include the amount of trypsin used for activation
as well as the time and temperature of incubation. In
the dog, an inactive renin-like enzyme has been reported to increase after nephrectomy.34"36 In the experiments of Gallagher et al.34 and Wilczynski and Osmond,36 the increase in inactive renin was
demonstrated by using a pH in the range of 5.7 to 6.0
for the ANG I generation. Gallagher et al.34 suggested
that the increase in activity did not appear to result
from an increase in the concentration of renin, since
the optimum pH of angiotensin formation in trypsintreated plasma from anephric dogs was pH 5 or less.
They concluded that the trypsin activatable renin-like
enzyme was cathepsin D.
In our present study, we have shown a remarkable
increase of rat inactive renin after bilateral nephrectomy. In our study, inactive renin has a molecular
weight of approximately 60,000, and its pH optimum
is between 5.5 and 7.5, which is different from that of
cathepsin D and pepsin. Also, inactive renin was capable of generating ANG I when natural renin substrate
was used at pH 7.4. Cathepsin D usually cannot act on
natural renin substrate, but trypsin has been reported to
have generated tetradecapeptide from natural renin
substrate in horse plasma.37 Therefore, cathepsin D
could possibly act upon the tetradecapeptide cleaved
by trypsin from the natural substrate. However, the pH
optimum should be lower and no ANG I should be
generated at pH 7.4. Also, if this were the case, one
631
might expect that the rate of ANG I generation after
nephrectomy would be proportional to the renin substrate concentration. Our data did not show a direct
relationship with substrate concentration, because the
maximum value of inactive renin was at 20 hours and
then gradually decreased, while substrate concentration after nephrectomy peaks at between 8 and 24
hours and remains elevated for at least 48 hours.30' "• 38
Furthermore, after elution from Sephacryl S-200 and
Cibacron blue, activated inactive renin generates ANG
I from natural substrate that had never been exposed to
trypsin. Finally, under our experimental conditions,
trypsin did not produce any substance that interfered in
the RIA for ANG I. If tetradecapeptide were generated, one might expect cross-reactivity in the RIA.
These observations suggest that in the rat inactive renin
present after nephrectomy is not cathepsin D.
The molecular weight of inactive renin after nephrectomy in the rat has not been reported previously.
In this study, we showed that inactive renin has a
molecular weight of approximately 60,000. It does not
change after nephrectomy, and it does not change after
trypsin activation.
The source of plasma inactive renin is still controversial. Although inactive renin has been found in human and hog kidneys,22-23 the ratio of inactive-to-active renin is lower in the kidney than in the peripheral
plasma. These data suggest that, in addition to the
kidney, there is an extrarenal source of inactive renin.
Furthermore, inactive renin has been found in humans
after bilateral nephrectomy, which indicates that the
kidney is not the only source of inactive renin. 2426
Weinberger et al.24 suggested that the source of inactive renin after nephrectomy was the salivary gland. In
our study, however, the salivary gland did not appear
to be the only source of inactive renin in the rat, since
the rise of inactive renin 20 hours after nephrectomy
was not prevented by the simultaneous removal of the
salivary glands. Furthermore, removal of the adrenals,
pituitary, uterus, pancreas, stomach, and intestines did
not prevent the rise of inactive renin. It is possible that
other organs such as the brain, liver, lungs, or the
vascular endothelium may be the source of this enzyme. The physiological role and mechanism of the
secretion of inactive renin are still unknown. In our
experiment, inactive renin increased markedly after
nephrectomy. Sodium loading and ANG II did not
prevent the increase of inactive renin after nephrectomy. In contrast to active renin, these data suggest
that the secretion of inactive renin is not controlled by
volume or negative feedback by ANG II.
In summary, we confirmed an enzyme in the plasma
of nephrectomized rats with the characteristics of inactive renin. We were unable to determine the tissue of
origin nor its physiological role, but this enzyme is not
converted to active renin in peripheral blood. Furthermore, any changes in the concentration of inactive
renin in peripheral blood of rats cannot be ascribed to
changes in renal secretion unless arteriovenous differences across the kidney are measured.
HYPERTENSION
632
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Y Doi, R Franco-Saenz and P J Mulrow
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Hypertension. 1984;6:627-632
doi: 10.1161/01.HYP.6.5.627
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