The Storage Form of Renin in Renin Granules
from Rat Kidney Cortex
MINORU KAWAMURA, JAMES C. M C K E N Z I E , LOREN H.
HOFFMAN,
ISSEI TANAKA, M A R C PARMENTIER, AND TADASHI INAGAMI
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SUMMARY Renin granules were partially purified from rat kidney cortex, and a storage form of
renin in the granules was examined. Renin granules were isolated by discontinuous Per coll density
gradient centrifugation followed by continuous Percoll density gradient centrifugation. The partially
purified fraction was free from mitochondria and microsomes, as judged by the absence of marker
enzymes of these organdies, but contained some lysosomal enzyme activities. The specific renin
activity was 0.58 mg angiotensin I/hr/mg protein, 500 times as active as the original homogenate.
Immunochemical staining with specific antisera against rat kidney renin revealed that about 10% of
the granules recovered in the partially purified fractions were stained strongly. The stored renin was
not activated either by acidification or by trypsin treatment, indicating that stored renin was in the
fully active form. By sodium dodecyl sulfate gel electrophoresis, the stored renin had two different
molecular weights, 38,000 and 36,000, and these molecular weights were not reduced by dithiothreitol
or 2-mercaptoethanol, suggesting that these renins are single-chain types as opposed to the two-chain
type found in male mouse submaxillary gland. These results suggest that active renins with two
different molecular weights may be released from renin granules of juxtaglomerular cells.
(Hypertension 8: 706-711, 1986)
KEY WORDS • renin granules • purification
Percoll density gradient centrifugation
T
HE secretion of renin from juxtaglomerular
cells is regulated by a variety of stimuli that
presumably are transmitted by the baroreceptor, the neurogenic receptor, and the macula densa
receptor.' It is generally accepted that calcium acts as a
negative modulator of renin secretion,2 as it does in the
release of parathyroid hormone. Although much evidence indicates that renin is stored in specific granules
of juxtaglomerular cells, 3 little is known about the
renin secretory process because of the extremely low
number of juxtaglomerular cells in the kidney. Renin
constitutes only 0.01% of kidney proteins. 4
Recently, the presence of more than one pathway of
renin secretion has been suggested by several groups of
investigators.5"8 In one pathway, renin is stored in
granules before release, while in another pathway
rat renin
• storage form
newly synthesized renin is secreted directly. To gain
further insight into the mechanism of the renin secretory process, information must be obtained on the biochemical nature of the stored renin. In most studies,
renin granules have been isolated by a discontinuous
sucrose density gradient centrifugation method from
human,9 dog,10 rabbit," and rat12 kidneys. The specific
activity of renin granule preparation was enriched at
most five to 20 times as compared with that of the
homogenate of the whole kidney cortex. We have attempted to obtain a higher degree of purification by
devising a different procedure. Thus, the goal of the
present report was to isolate renin granules using this
new method and to study the storage forms of renin.
Materials and Methods
Animals and Fractionation of Homogenate
Male Sprague-Dawley rats, weighing 180 to 220 g,
were fed Purina rat chow (St. Louis, MO, USA) containing 0.39% sodium and allowed free access to tap
water. Rats were anesthetized with sodium pentobarbital (5 mg/100 g body weight i.p.), and an abdominal
incision was made to expose the kidneys. Kidneys
were removed after perfusion with isotonic saline,13
and the kidney cortex was minced and gently homog-
From the Department of Biochemistry and Hypertension Center,
Vanderbilt University School of Medicine, Nashville, Tennessee.
Supported by National Institutes of Health Grant HL-14192. Dr.
McKenzie was a postdoctoral hypertension trainee supported by
National Institutes of Health Training Grant HL 07323. Dr. Parmentier was a Fogarty International Fellow.
Address for reprints: Tadashi Inagami, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN
37232.
Submitted July 22, 1985; accepted February 5, 1986.
706
RAT KIDNEY RENIN GRANULES/Kawamura et al.
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enized with 0.45 M sucrose (1:7 wt/vol).9 The homogenate was centrifuged at 500 g for 10 minutes, and 4 ml
of the supernatant (original homogenate) then was
placed on a discontinuous Percoll density gradient solution (Sigma Chemical, St. Louis, MO, USA). The
Percoll solution was made isosmotic with 0.25 M sucrose (final concentration). The gradient consisted of
three 8-ml layers of 1.07, 1.11, and 1.15 g/ml Percoll
solutions. After centrifugation at 40,000 g for 15 minutes, the gradient had three visible bands. The gradient
was separated into four fractions, F0 (volume, 4.0 ml),
Fl (4.0 ml), F2 (7.0 ml), and F3 (11 ml), from the top
of the layers of the solution by using an ISCO Model
185 Fractionater (Instrumentation Specialties, Lincoln, NE, USA). Then, 10 ml of F3 solution was
added to a polycarbonate tube (1.4 X 13 cm), centrifuged at 20,000 g for 90 minutes using a JA-20 rotor
(Beckman, Palo Alto, CA, USA), and fractionated
into 10 fractions (1 ml each) by pipetting. Renin granules were lysed by mixing the granule-containing solution with 0.1 volume of phosphate buffer (0.1 M, pH
7.0) containing 1% Triton X-100. The mixture was
centrifuged at 100,000 g for 120 minutes, and the
supernatant was used for renin activity assay. Density
was calculated from refractive index using an Abbe
refractometer (Fisher Scientific, Pittsburgh, PA,
USA).
Estimation of Molecular Weight
Gel filtration was performed on a Sephadex G-75
column (Pharmacia, Piscataway, NJ, USA); 1 ml of a
diluted sample containing molecular weight markers
was applied to a Sephadex G-75 column (1.6 X 90 cm)
that had been equilibrated with phosphate buffer (50
rhM, ph 7.2) containing 0.1 M NaCl, and 1 ml fractions were collected for measurement of renin activity.
The molecular weight of renin was estimated by
sodium dodecyl sulfate gel electrophoresis followed
by the Western blotting technique as detailed by Burnette.14 Samples were electrophoresed in 11% sodium
dodecyl sulfate-polyacrylamide gel according to the
method of Laemmli.15 The proteins were transferred
electrophoretically to a nitrocellulose sheet (5 hours at
3 V/cm in 40 mM Na phosphate buffer, pH 6.5). The
sheet was treated with anti-rat kidney renin antibody16
at a 1:500 dilution, washed thoroughly, then treated
with radioiodinated protein A (70-100 /nCi//ig; New
England Nuclear, Boston, MA, USA) at a concentration of 5 x 103 cpm/ml. After extensive washing, the
sheet was exposed at - 7 0 ° C to Kodak XAR5 film
(Rochester, NY, USA). Before application to the electrophoresis gel, the sample was divided into three portions and pretreated as follows. The first sample was
heated for 3 minutes at 90 °C and then centrifuged at
13,000 g for 5 minutes. The supernatant then was
collected for the electrophoresis. The second sample
was heated to 90°C in the presence of 5% 2-mercaptoethanol and then was treated in the same way as the
first sample. The third sample was incubated at 25 °C
for 1 hour in the presence of 10 mM dithiothreitol. The
sample was then filtered through a Diafilter membrane
707
YM 10 (Amicon, Danvers, MA, USA) with 50 volumes of tris(hydroxymethyl) aminomethane(Tris) HC1
buffer (20 mM, pH 7.5) containing 1 mM dithiothreitol. The dithiothreitol-treated sample was then
treated with mercaptoethanol in the same way as the
second sample.
Measurement of Enzyme Activities and Protein
Content
Renin activity was measured by the rate of formation of angiotensin I (ANG I) from rat angiotensinogen
as described previously.13 The ANG I produced was
assayed by radioimmunoassay according to the method of Haber et al. l7 Succinate dehydrogenase, a marker
enzyme of mitochondria, was assayed by the method
of Slater and Bonner.18 Glucose-6-phosphatase, a
marker for microsomes, and acid phosphatase, a lysosomal marker, were assayed according to the method
of Morimoto et al. l9 Acid protease, another lysosomal
marker, was determined by a modification* of the
method of Williams and Lin.20 In preparatory experiments, we confirmed that these enzyme activities were
not influenced by the addition of Percoll solutions under conditions used in the present assay system. The
protein concentration was determined by the method of
Lowry et al., 21 using bovine serum albumin as the
standard. As Percoll caused a background coloration
with the reagent for the protein assay, each sample
value was corrected for background obtained with a
Percoll solution of an identical concentration not containing the kidney homogenate.
Activation of Inactive Renin
Activation of inactive renin was performed separately by acidification and by trypsin treatment. Acidification was performed as reported previously. 9 Samples were diluted with 4 volumes of phosphate buffer
(50 mM, pH 7.0) containing 0.1 M NaCl. Then, 0.5
ml of sample was dialyzed against glycine HC1 (50
mM, pH 3.3) containing 0.1 M NaCl for 24 hours and
subsequently against phosphate buffer (50 mM, pH
7.0) containing 0.1 M NaCl for 24 hours. As a control,
the samples were dialyzed against the neutral buffer for
48 hours. Trypsin treatment was performed as reported
previously.22 To remove active renin, 5 ml of sample
was applied to a pepstatin Sepharose column. The
pass-through fraction was concentrated to 5 ml, and
the aliquot was incubated with trypsin (100 /Ag/ml) up
to 60 minutes. After the reaction was stopped by the
addition of soybean trypsin inhibitor, the renin activity
of each sample was measured.
Immunocytochemistry
For immunocytochemistry analyses, 20 volumes of
0.45 M sucrose was added to a fractionated sample and
the solution was centrifuged at 5,000 g for 10 minutes.
The pellets were fixed for 3 hours at 25 °C in phosphate
buffer (0.1 M, pH 7.4) containing 4% paraformaldehyde, 0.25% glutaraldehyde, and 0.3% picric acid.
The pellets were then dehydrated in dimethyl formamide and embedded in Lowicryl K 4M (Polysciences,
708
HYPERTENSION
VOL
8, No 8, AUGUST 1986
TABLE 1. Enzyme and Protein Distribution in Fractions After Discontinuous Percoll Density Gradient Centrifugation
Fractions isolated by Percoll density gradient
Original
homogenate
F0
F2
Variable
Fl
F3
95.7±5.63
33.5±2.48
5.6±0.42
7.63±O.7O
50.6±4.73
Renin (/tg ANG I/hr)
584+13.2
102±8.50
151 ±12.1
153±11.9
32.8±2.20
Acid protease (103 cpm/hr)
350 + 32.7
33.4±3.06
Acid phosphatase*
1580±88.3
779±53.9
424±34.5
Glucose-6-phosphatase*
883 + 21.6
246±9.87
594±37.1
9.59±0.29
0
Succinate dehydrogenaset
266±16.5
19.2±2.60
52.4±6.4
137±13.9
0
Protein (mg)
81.7±0.99
36.2±1.40
31.1 ±1.30
14.2 + 0.30
2.20±0.20
Values are means ± SE offiveexperiments. Enzyme activities and protein contents in 4 ml of original homogenate and
in each fraction volume. F = fraction; ANG I = angiotensin I.
•Measured as micrograms of released inorganic phosphate from substrate per hour.
tMeasured as decrease of absorbancy at 400 nm/hr.
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Warrington, PA, USA). The gel was polymerized by
exposure to ultraviolet light. Sections were cut 65 nm
thick with an LKB in ultramicrotome (Bromma,
Sweden) and mounted on 200-mesh nickel grids. The
grids were then immunostained using the immunogold
technique as reviewed by Horisberger.23 Sections were
incubated for 20 hours at 4°C with a 1:100 dilution of
anti-renin serum. The grids were thoroughly washed
and incubated for 1 hour with goat anti-rabbit IgG-5
nm gold at a dilution of 1:50. Following stabilization
of 1% glutaraldehyde and staining with 1% uranyl
acetate, the sections were examined in a Hitachi H-600
electron microscope (Hitachi, Tokyo, Japan). Normal
rabbit serum was substituted for primary antibody as a
control.
Results
Renin granules were isolated by discontinuous and
then continuous Percoll density gradient centrifugation. Table 1 and Figure 1 show the average distributions of marker enzymes and proteins in each fraction
separated by the discontinuous centrifugation. Approximately 53% of the total renin activity was contained in F3 (interphase between 1.11 and 1.15 g/ml).
•
Acid ProttoM
C D Acid Phosphotou
EDffrnto
•
Prortin
Glucose-6-phosphatase and succinate dehydrogenase
activities were not detected, while 5% of the total
lysosomal marker enzyme activity was in this fraction.
The specific activity of renin in F3 was 23.0 fig ANG
I/hr/mg protein, which was 20 times that of the original homogenate. Figure 2 shows enzyme activities
and protein concentration of fractionated samples
after continuous Percoll density gradient centrifugation of 10 ml of F3. Renin activity was found to peak
in Fraction 8 (partially purified renin granule fraction), which corresponded to a density of 1.138 g/ml.
The specific activity of renin in this fraction was
0.58 ± 0.08 mg ANG I/hr/mg protein, which was 500
times that of the original homogenate. Of the total
renin activity in F3 applied to the continuous gradient,
24% was present in Fraction 8. Thus, 13% of the total
renin activity in the original homogenate was recovered in this peak fraction. This figure contrasts with an
overall recovery of 0.4% of acid protease activity in
Fraction 8.
The partially purified fraction consisted of round
and oval granules 0.4 to 1.5 /xm in diameter. There
was no contamination by mitochondria (Figure 3A).
When this sample was stained by the renin-antibodyimmunogold technique, all granules were stained posi-
d Sucdnds Dehydroganase
CD Glucose S-PtKnphatae
TO
F1
rap
20
4O
60
FIGURE 1. Distribution patterns of enzymes and protein in
fractions obtained by discontinuous Percoll density gradient
centrifugation. Abscissa shows distribution of marker enzyme
activities (fraction volume x enzyme activity) and protein contents (fraction volume x protein concentration) in the fractions
isolated as a percentage of the total recovered in all fractions
(F0 + Fl + F2 + F3). Ordinate shows fraction numbers.
These data were calculated from data in Table 1.
2 3 4 5 6 7 8 9
10
FRACTION NUMBER
FIGURE 2. Distribution patterns of enzyme activity and protein concentration obtained after a continuous Percoll density
gradient centrifugation of Fraction 3 (F3) obtained from the
discontinuous gradient centrifugation. Values are means ±SE
of five experiments.
RAT KIDNEY RENIN GRANULES/Kawatmira et al.
709
•"<•:•
.-•
, T.
-»
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FIGURE 3. Electron micrograph of subcellular organelles recovered in the partially purified fraction (Fraction
8). A. Renin granules were stained only with uranyl acetate (magnification x 19,000). No contamination of
mitochondria can be seen. B. Granules were stained with renin-antibody and gold-labeled goat anti-rabbit IgG
(magnification x 55,000).
tively to a varying extent. Approximately 10% of granules were stained very heavily, while the remaining
granules were lightly stained (Figure 3B). In control
samples, normal serum used in place of anti-renin antibodies failed to stain the organelles (data not shown).
The storage form of renin was examined using the
partially purified renin granule fraction. When the
renin from the fraction was treated by acid dialysis, no
change in renin activity (mean ± SE, 3.41 ± 0.41 vs
3.42 ± 0.39 fig ANG I/hr/ml; n = 5) was observed.
No renin activity appeared after trypsin treatment of
the flow-through fraction (active renin-free fraction)
obtained from a pepstatin column to which the renin
from the partially purified fraction was applied. When
renin extracted from the granules was applied to a
Sephadex G-75 column, a single peak of renin activity
emerged in the position corresponding to a molecular
weight of 38,000 (Figure 4). The use of sodium dodecyl sulfate gel electrophoresis followed by Western
blotting elicited two bands at molecular weights of
38,000 and 36,000. After treatment with 2-mercaptoethanol, or dithiothreitol, or both, no change in the
molecular weight of these bands was noted (Figure 5).
Discussion
Renin secretion is known to be proportional, to
some extent, to the renal renin content, 5 and the content is correlated with the number of renin granules in
the juxtaglomerular cells.24' ^ The storage of renin in
renin granules seems to occur in the main pathway to
Bovine serum albumin Ovalbumln
oC-Chymotrypsinogen A
e ioo-
70
80
90
100
Fraction Number
FIGURE 4. Gel filtration of the partially purified fraction
(Fraction 8) on Sephadex G-75 column. Al = angiotensin I.
renin secretion. Because renin has been reported to
exist in multiple molecular weight forms in male
mouse submaxillary gland8> M and kidney,27 determination of the biochemical nature of renin in renin granules is an approach to the clarification of the renin
secretory process.
Although various methods, such as free-flow electrophoresis,28 a sieving method,29 and sucrose density
gradient centrifugation,9-12 have been employed for the
isolation of renin granules, these methods have not
HYPERTENSION
710
1X3
Bovine serum albumin
-»
Ovalbumin ->
Ml
Carbonic onhydraw
-»
FIOURE 5. Autoradiograph of 11% sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Lane 1. No treatment of
the partially purified fraction (Fraction 8). Lane 2. 2-Mercaptoethanol treatment of the partially purifiedfraction.
Lane
3. 2-Me'rcaptoetharwl and dithiothreitol treatment of the partially purified fraction. Details of separation conditions and
treatment with thiol reductants are given in Methods.
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produced completely satisfactory results. In the present study, we applied a Percoll density gradient centrifugation for the isolation of renin granules and obtained a 500-fold purification in specific renin activity
and 14% in recovery over the original homogenate.
Thus, this method appears to be superior in purity and
convenience to the previous methods. There are several reasons for the increased purity of renin granules
achieved by this method. First, a primary renin granule
fraction (F3) can be obtained quickly. As Percoll has
low viscosity, renin granules sediment to an equilibrium position in the gradient in 15 minutes at 40,000 g as
compared with 90 minutes at 60,000 g in a sucrose
density gradient. Second, the primary granule fraction
can be quickly recentrifuged for a further separation by
a continuous Percoll density gradient. Third, during
the isolating procedures, renin granules are kept in
isosmotic conditions. Thus, the prevention of osmotic
shock, as well as physical shock resulting from prolonged manipulation of the pellet, reduces damage to
the granule during purification.
When the purity of the partially purified renin granule fraction was examined by electron microscopy, all
granules were stained positively for renin. No granules
were stained in controls using normal rabbit sera.
Thus, all granules that were recovered in the fraction
seem to contain renin, even in small quantities. The
complete purification of renin from rat kidney requires
a 6000-fold to 8000-fold purification. In the present
work, renin activity was enriched 500-fold in the partially purified renin granules. Thus, it is in agreement
with the present finding that approximately 10% of the
granules constitute the major storage vesicles of renin.
These intensely stained renin granules containing renin
at a very high concentration may be considered the
"mature" renin granules proposed by Barajas.30 He has
also described protogranules (immature granules),
which are 0.09 to 0.4 ^im in diameter and round or
diamond shaped. On the other hand, approximately
90% of the granules seem to contain only small quantities of renin, as indicated by the low density of gold
particles in the electron microscopic studies. The present preparation of renin granules contained some lysosomal enzyme activity. Earlier reports31-32 indicated
VOL 8,
No
8,
AUGUST
1986
the coexistence of renin and lysosomal enzymes in
what the authors called "renin granules." Thus, the
likelihood that granules containing lower concentrations ofreninmay have mixed properties of renin granules and lysosomes cannot be eliminated.
The renin in the partially purified fraction from rat
kidney was in the active form. On the other hand, we
found that inactive renin constitutes 20% of total renin
activity (active renin + inactive renin) in human renin
granules9 and 0.3% in dog renin granules.22 We also
found inactive renin in microsomal fractions in rat
kidney (unpublished observation). Thus, conversion
of inactive to active renin during maturation of renin
granules appears to be more rapid in dogs and rats than
in humans.31 The molecular weight of the stored renin
showed one peak in the vicinity of 40,000 by gel chromatography. This observation is in agreement with
those reported from other laboratories.12'33 By sodium
dodecyl sulfate gel electrophoresis, however, two different molecular weights (38,000 and 36,000) of renin
were found in the granule fraction in the present studies. Purified rat kidney renin was also found to exist in
these two molecular weights.4 In contrast to the active
renin, the inactive renin of rat kidney has been shown
to have a molecular weight of 48,000 by gel electrophoresis. 16 These results are all in agreement with the
concept that the 36,000 and 38,000 (molecular weight)
renins in the present renin granule preparation are of
the active form.
Pratt et al.8 indicated that granules of the mouse
submandibular gland contain two-chain renin that consists of a heavy chain and a light chain linked by a
disulfide bridge, as was found in purified mouse submandibular gldnd renin.34 However, the molecular
weights of stored renin in rat kidney were not changed
by treatment with thiol reductant. These results suggest that the stored renins in the rat kidney are of the
one-chain type. We cannot exclude the possibility that
renin with a molecular weight of 36,000 could be an
artifact generated during the isolation procedures. Further studies are needed to determine whether these two
forms of renin are released from the juxtaglomerular
cells or from different sources.
In conclusion, the results reported herein indicate
that active renin of two different molecular weights is
stored in renin granules of the rat kidney and is secreted from mature renin granules.
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M Kawamura, J C McKenzie, L H Hoffman, I Tanaka, M Parmentier and T Inagami
Hypertension. 1986;8:706-711
doi: 10.1161/01.HYP.8.8.706
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