Kallikrein and Prekallikrein on the
Basolateral Membrane of Rat Kidney Tubules
KENICHI YAMADA, M.D.,
WERNER W. SCHULZ, P H . D . , DEBBIE S. PAGE,
B.S.,
AND ERVIN G. ERDOS
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SUMMARY Basolateral membrane (BLM) enriched fraction was isolated from homogenized rat kidney
cortex by differential centrifugation. We also obtained a fraction enriched in plasma membrane (PM). The
morphology of the Isolated BLM fragments was studied by transmission and freeze fracture electron microscopy. The relative specific activity of Na + -K + -ATPase was enriched 7-fold, while that of marker enzymes for
PM, endoplasmic reticulum, and lysosomes was lower than In the crude bomogenate. There was a 10-fold
difference in the ratios of activities of Na + -K + -ATPase to Mg*+-ATPase in the BLM and in the PM enriched
fractions. Kallikrein activity was determined with S-2266 substrate and by radioimmunoassay of kinin released. It was low In the BLM fraction prior to adding detergent, but Triton X-100 increased the activity 12 to
16-fold. Both free trypsin and Sepharose 4B-bound insoluble trypsin Increased kallikrein activity 2- to Mold in
both the membrane-bound and soluble fractions, probably by activating a prekallikrein. The results were Interpreted that the kallikrein studied originated from the distal tubular BLM.
(Hypertension 3 (suppl II): II-59-II-64, 1981)
KEY WORDS • kininogenase • kinlns
• renal enzymes • Na + K + ATPase
• prekallikrein activation
R
•
kallikrein release
ENAL distribution of enzymes that release
or inactivate kinins has been studied by a
number of investigators.1' * The glomerulus
has little or no kallikrein and kininase II activity.*
Most, but not all, of kininase II or converting enzyme
is concentrated on the brush border of the proximal
tubules.*""* Urinary kallikrein enters the nephron at the
level of the distal tubules.1 Kallikrein in the kidney is
bound to plasma membrane, as shown by the isolation membrane fractions enriched in kallikrein1 and
by the kallikrein activity expressed on the surface of
isolated, suspended kidney cells.8 Immunofluorescence studies first indicated that kallikrein is located
on the luminal side of the distal part of the nephron,*
but recently it was suggested that kallikrein may be
more uniformly distributed in these cells. 7 ' Kallikrein is released by vasoactive agents given into the
renal artery,10 and appears in the renal lymph and
venous effluent.11 It was suggested that renal kallikrein affects renal vascular resistance." These findings are hard to reconcile with a primary localization
of kallikrein on the luminal side of the nephron.
Because of the reported discrepancies in functions,
release and localization of renal kallikrein, we isolated a basolateral membrane (BLM) enriched fraction from the rat kidney and studied enzymes in this
fraction. This communication describes the technique
used for the separation of a BLM enriched fraction
containing bound prekallikrein and kallikrein. The
properties of the BLM-bound kallikrein were compared with those of a kallikrein in the plasma membrane (PM) enriched fraction.1'"18
From the Departments of Pharmacology and Internal Medicine
and Pathology, University of Texas Health Science Center, Dallas,
Texas.
Supported in part by Grants HL 20594-04, HL 14187-10, and HL
16320-06 from the U S. Public Health Service, National Institutes
of Health.
Address for reprints. Dr. E. G ErdSs, Department of Pharmacology, University of Texas Health Science Center, Dallas, Texas
75235
Fractlonation (Table 1)
Material and Methods
D-Val-Leu-Arg-p-nitroanilide (S-2266) was purchased from Kabi AB, Stockholm; trypsin from
Worthington Biochemicals Corporation, Freehold,
New Jersey; soybean trypsin inhibitor from Sigma
Chemical, St. Louis, Missouri; CNBr-activated
Sepharose 4B from Pharmacia Chemicals, Piscataway, New Jersey. Other chemicals were obtained from
Eastman Kodak Company, Rochester, New York.
Antiserum to bradykinin was donated by Dr. F.
Alhenc-Gelas, Paris, France.
Male Sprague-Dawley rats (200-250 g) were killed
by decapitation. All subsequent steps were carried out
at 4°C. Kidney cortex was separated from medulla
and dissected into small pieces. These pieces were
gently homogenized with a Teflon homogenizer in 1:5
(w/w) buffer (5 mM Tris-HCl 7.4, containing 0.5 mM
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TABLE 1. Experimental Steps
Rat kidney cortex
Homog. in Teflon homog. 1:6 (w/w) in 5 mM Tris pH 7.4, 0.5 mM CaCla, 0.25 M sucrose
I
200 g X 5 minutes
ppt
sup.
filtered 180 Teflon mesh filter
I
2,000 g 20 minutes; resuspended, washed, and recentrifuged 10 times
sup
ppt
Ucfg 90,000 g, 2 hours
Discontinuous sucrose density gradient in Tris-CaCla buffer
1.16 < d < 1.18 (g/ml)
repeat step with d = 1.22 buffered sucrose added
I
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cfg. at 22,000
2,00 g for 20 minutes in homogenate buffer
/ \\ \
sup
ppt — wash with 0.25 M sucrose, 10 mM Tris pH 7.4, repeat step twice
Basolateral membrane enriched fraction
I
Resuspend in hypotonic 10 mM. Tris pH 8.6, 0.5 mM CaCU, and cfg.
100,000 g, 45 minutes; repeat with 1 mM Tris, pH 8.6, 0.5 mM CaCU
Wash ppt with 0.25 M sucrose + 10 mM Tris, pH 7.4 and cfg. 22,000 g 20
min; repeat step once
Washed basolateral membrane fraction
CaClt and 0.25 M sucrose). After brief centrifugation
(200 X g for 5 min), the resultant supernatant was
filtered through 180 Teflon mesh. The filtrate was then
centrifuged at 2,000 X g for 20 minutes. The pellet
was resuspended in the homogenate buffer, washed,
and recentrifuged 10 times. The final precipitate was
suspended in homogenate buffer. This fraction was
centrifuged at 90,000 X g for 2 hours in a Beckman
SW 27 rotor of the ultracentrifuge over a discontinuous density gradient consisting of 1.16, 1.18 and 1.20
g/ml, sucrose, in 5 mM Tris HC1, pH 7.4, 0.5 mM
CaCl,. The layer at the interface of the 1.16 and 1.18
g/ml sucrose solutions was collected. Then the density of the bottom fraction was adjusted to 1.22 g/ml
with the addition of 65% sucrose, 5 mM Tris HC1, pH
7.4, 0.5 mM CaClj. The centrifugation was repeated
at 90,000 X g for 2 hours, and at the layer on the interface of 1.16 and 1.18 g/ml sucrose solution was collected. After dilution to isotonicity with 5 mM Tris
HC1, pH 7.4, and 0.5 mM CaCl,, the membrane fraction was sedimented at 22,000 X g in 20 minutes. The
pellet was suspended in 10 mM Tris HC1, pH 7.4 and
0.25M sucrose, washed and recentrifuged at 22,000 X
g for 20 minutes. The resultant pellet, the BLM rich
fraction, was suspended in 10 mM Tris HC1 pH 7.4,
containing 0.25 M sucrose. For the hypotonic washing, the fraction was suspended in 10 mM Tris HC1,
pH 8.6, and 0.5 mM CaCl,, washed, and centrifuged
at 100,000 X g for 45 minutes. It was then suspended
in 1 mM Tris HC1, pH 8.6 containing 0.5 mM CaCla,
and centrifuged at 100,000 X g for 45 minutes. This
step was followed by washing twice with 10 mM Tris
HC1 pH 7.4 and 0.25 M sucrose and centrifugation at
22,000 X g for 20 minutes. The final washed BLM
pellet was suspended in 10 mM Tris HC1, pH 7.4 containing 0.25 M sucrose.1*- " Washed basolateral membrane was solubilized by incubation in 0.1% Triton X100 for 30 minutes at 4°C and centrifuged at 105,000
X g for 60 minutes.
Assays
The following marker enzymes were assayed as
reported previously:1-14 5'-nucleotidase, Na-KATPase, Mg-ATPase, alkaline phosphatase, acid
phosphatase, glucose-6-phosphatase, j8-glucuronidase,
and aminopeptidase A. The substrates were: adenosine-5-monophospate, ATP Na,, p-nitrophenyl
phosphate, glucose-6-phosphate, p-nitrophenyl glucuronide, and aspartyl-0-naphthylamide respectively.
The activity of kallikrein was assayed with S22661*"1* and by determining the kinin released from
heated dog plasma by radioimmunoassay. The incubation mixture contained 0.1M Tris HC1, pH 8.5,
and kininase inhibitors SQ 14,225, (captopril 10"* M),
EDTA (5 X 10"' M) and o-phenanthroline (10"' M).
Sepharose-4B-bound trypsin was prepared by using
Sepharose 4B activated by CNBr according to the
manufacturer's recommended procedure. Approximately 75% of trypsin was bound to gel and approximately 20% of the activity of trypsin was retained. The
activity of trypsin was measured by using S-2266 as
11-61
KALLIKREIN IN BASOLATERAL MEMBRANE/Yamada et al.
substrate at pH 9.0. Sepharose 4B-bound trypsin was
suspended in 0.1 M sodium acetate buffer, pH 4.0,
containing 1M NaCl and Na azide and stored at 4°C.
An aliquot of washed or solubilized BLM was treated
with trypsin by mixing 10 ng of free trypsin or
equivalent activity of bound trypsin and incubating at
room temperature for 60 minutes in 10 mM Tris at
pH 7.4 in tubes rotated on a turntable. After incubation with soluble trypsin, 20 jtg of soybean trypsin inhibitor was added to stop the reaction. This amount of
inhibitor completely inhibited all trypsin activity.
Samples with bound trypsin were immediately centrifuged at 250 X g for 3 minutes, and the supematants
were removed and used in assays. No trypsin activity
was detected in the supernatants after Sepharose-4B
bound trypsin was sedimented.
Electron Microscopy
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The pellet of basolateral plasma membrane was
fixed in 3% glutaraldehyde for several days and
postfixed in 1% osmium tetroxide for 1 hour. Both fixatives contained 0.1 M phosphate buffer and 5%
sucrose, pH 7.3. Following dehydration through
graded alcohol and propylene oxide, the sample was
embedded in Epon-Araldite." Thin sections were
stained and sectioned for electronmicroscopy, with
uranyl acetate, and lead citrate. They were observed
and photographed in the JEOL 100S electron microscope.
Freeze-fracture replication was executed in a Denton DFE-3 freeze-etch module. Tissue from the cortex
of rat kidney and BLM pellet were fixed in 3%
glutaraldehyde for several days. For cryoprotection,
specimens were placed overnight in 30% glycerol containing 1% glutaraldehyde. The specimens were
loaded into apposed-specimen gold holders and
dropped into Freon 22 at — 165°C. They were fractured at -105°C and slightly etched while the
temperature was allowed to rise to — 100°C. Platinum
shadowing was carried out using an electron beam gun
of our own design.1* Replicas were cleaned in commercial bleach containing 1.3% sodium hypochlorite and
rinsed three times in distilled water. The replicas were
photographed in the JEOL 100C electron microscope.
The electron microscope sheet film was reversal
processed so that shadows appear black on the
prints.40
The details of separating plasma membrane (PM)
and endoplasmic reticulum (ER)1*"16 enriched fractions from the homogenized rat kidney cortex were
given in the previous communications from this
laboratory.
Results
Marker Enzymes
Table 2 summarizes the distribution of marker enzymes in the fractions isolated from homogenized rat
kidney cortex. Fraction II BLM had only 2.5% of the
total protein but the relative specific activity of Na + K + -ATPase increased sevenfold, with a yield of 18%.
Repeated washing did not change the activity of the
enzyme in the BLM fraction. In contrast, the relative
specific activity of alkaline phosphatase, a PM marker
enzyme and that of glucose-6-phosphatase, an ER
marker, decreased in the BLM fraction. Aminopeptidase A activity also decreased, while 5'-nucleotidase, a membrane-bound enzyme, was enriched 2.4fold. The relative specific activities of acid
phosphatase, and /3-glucuronidase, lysosomal markers
decreased from 1 to 0.3 in the washed BLM fraction
(not shown).
Electronmicroscopy
Transmission electron microscopy revealed that the
BLM fraction consisted of vesicles ranging from 0.2 to
2.0 nm in size (fig. 1). While the majority of vesicles
appeared empty, some contained smaller vesicles.
These smaller vesicles may have been trapped as the
BLM broke apart and vesiculated during homogenization. In freeze-fracture images large membrane sheets
were seen (fig. 2), which appeared to form multiple
sheaths around each other. This arrangement is very
similar to the basal plasma membrane infoldings seen
in distal tubules in situ (fig. 3). Occasionally, both in
transmission electron microscopy and in freeze-frac-
TABLE 2. Marker Enzymes in Subcellular Fractions of Rat Kidney Cortex
Na+-K+ATPase*
(jimole/
min/mg)
SA
RSA
Alkaline
phosphatase
Homogenate
2,000 g ppt
Fraction I
0.14
0.24
0.73
Fraction II
(BLM 1.16 < d < 1.18)
Fraction III
(1.18 < d < 1.20)
Washed BLM
Fraction
5'-Nucleotidase
(nmole/
min/mg)
Aminopeptidase A
(nmole/
Protein
min/mg)
recovery
SA
RSA
min/mg)
SA
RSA
Glucose-6
phosphatase
(pinole/
min/mg)
SA
RSA
SA
RSA
1
1.7
5.2
0.22
0.10
0.22
1
0.45
1.0
0.15
0.2
0.19
1
1.3
1.3
4.5
7.8
9.9
1
1.7
2.2
2.9
1.3
1.6
1
0.4
0.6
100
43
1.0
0.98
7.0
0.10
0.45
0.1
0.7
12.4
2.8
2.1
07
2.5
0.1
0.7
0.04
0.2
0.04
0.3
7.6
1.7
0.4
0.14
5.4
0.97
7.0
0.10
0.45
0.09
0.6
10.8
2.4
2.4
0.8
2.0
BLM = basolateral membrane; SA = specific activity; RSA = relative specific activity (n = 3).
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TABLE 3. Distribution of Kallikrem Activity Among Fractions of the Homogenized Rat Kidney Cortex
Hydrolysis of S-2266
(nmole/min/mg)
+ Triton
(0.1%)
Fraction
Homogenate
2,000 g sediment
Fraction I
0.25
0.16
0.24
0.25
0.18
0.7
Fraction II, BLM
(1.16<d<1.18)
Fraction III
0.1
Washed Fraction
II, BLM
Percent
. activation
Yield by Triton
100
31
3
0
13
192
0.8
8
700
0.07
05
11
614
0.15
1.25
10
733
BLM = basolateral membrane, n = 5.
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FIGURE 1. Transmission electron micrograph of washed
basolateral membrane fraction.
ture, mitochondria were seen wrapped in membrane in
the fractions as in the folds of basal membrane in the
intact distal tubules.
Kallikrein
Table 3 shows the distribution of kallikrein activity
among the isolated renal fractions. As measured with
S-2266 substrate, about 30% of the kallikrein activity
in the homogenate was recovered in the washed 2,000
X g sediment. Triton X-100 did not increase the activity of kallikrein in the crude homogenate or in the first
sediment. However, it enhanced kallikrein activity to
8-fold in the BLM fraction, but less in the other frac-
FIGURE 2. Freeze-fracture image of isolated basolateral
membrane. Large membrane sheets reminiscent of basal infoldings are seen (Intramembrane particle aggregation is
probably due to phase separation which occurred during the
isolation procedure in the cold.)
tions isolated by differential centrifugation. The BLM
enriched fraction had about 10% of the total kallikrein activity of the rat kidney, after solubilization
with a detergent, but without Triton only about 0.91.2%. This indicates that it contains mainly inactive,
bound kallikrein.
Table 4 compares the results obtained by measuring kallikrein activity with S-2266 substrate and by
determining the amount of kinin released by radioimmunoassay. For the sake of comparison, the results
of experiments with kallikrein bound to PM fraction
are included in table 4. Triton X-100 solubilized kallikrcin since recentrifugation of BLM or PM fractions
at 105,000x g did not decrease the activity in the
FIGURE 3. Freeze-fracture image of infoldings of basolateral membrane in distal convoluted tubule of rat kidney in
situ. Intramembrane particles are numerous on the inner
halves of the plasma membrane.
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KALLIKREIN IN BASOLATERAL MEMBRANE/Yamada et al.
TABLE 4. Effect of Solubilization on Rat Renal Cortical
BLM and PM Kallikreins
S-2266
Kinin release*
(nmole/min/mg) (ng/min/mg)
BLM
0.15 ±0.03
0.69 ± 0.07
BLM + Triton (0.1%)
1.25 ±0.12
6.1 ± 0.82
BLM + Triton
1.85 ± 0.3
11.3 ±1.01
(105,000 £
supernatant)
PM
1.54 ±0.13
10.07 ± 1
5.79 ± 0.3
30.5 ± 1.9
PM + Triton (0.1%)
6.14 ± 0.4
39.2 ± 2.1
PM + Triton
(105,000 s
supernatant
BLM = basolateral membrane; PM = plasma membrane.
n = 5.
* = radioimmunoassay of kinin released.
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supernatant. The increase in the rate of hydrolysis of
the tripeptide substrate, S-2266, by Triton is in good
agreement with the results of radioimmunoassay.
While the BLM kallikrein activity increased after
detergent treatment 12.3 and 16.4-fold with either substrate, PM kallikrein activity was enhanced only
about four-fold under the same conditions. That the
BLM fraction contained prekallikrein was shown in
experiments where soluble trypsin or Sepharosebound trypsin was added to the incubation mixture.
Free or bound trypsin increased kallikrein activity
both in the membrane fraction and after solubilization. A discrepancy in the activation was noted only
when kallikrein activity was measured with S-2266
after free trypsin was added to the BLM fraction. This
disproportional increase in S-2266 hydrolysis by trypsin may be due to activation of a second enzyme in the
fraction that cleaves S-2266 but is not necessarily
identical with kallikrein. (table 5)
To differentiate PM from BLM enriched fraction,
we compared the ratios of activity of Na+K+-ATPase
to Mga+ATPase in the isolated BLM fraction and to
the ratios obtained in the PM- and ER-enriched fractions. While the ratio of activities in the washed BLM
fraction was 2.6, it was 0.22 in the PM fraction. This is
taken as an added evidence that the fraction was indeed enriched in basolateral membrane, since Na + K + ATPase is localized on the BLM membrane.21"28
Discussion
We report here that kallikrein is present in the
BLM-enriched fraction of the rat kidney, mainly in
inactive form. The 18 different iso- and hypotonic
washings and sedimentation procedures used should
preclude entrapment of kallikrein or an unspecific adsorption of soluble kallikrein on membrane vesicles.
Most of the kallikrein in the BLM fraction was
inactive, while in the PM fraction a substantial
amount of PM-kallikrein was in active form.1*"16
Bound kallikrein is activated by trypsin and Triton X100. However, PM kallikrein activity was increased
by Triton X-100 fourfold while BLM kallikrein activity was enhanced much more (tables 3 and 4). Kallikrein in the BLM fraction, prior to Triton treatment,
released about 0.7 ng of bradykinin per minute, but 24
ng after solubilization and activation. The activity in
the PM fraction was still higher, however, after a
detergent was added.
Additional data indicate that kallikrein in BLM
fraction is also inhibited by antiserum to urinary kallikrein. That BLM prekallikrein would originate from
a contaminating PM fraction is unlikely because
among others, the physical properties of activated
BLM prekallikrein are also different. (Yamada and
Erd6s, unpublished.) We assume that kallikrein in the
PM fraction came mostly from the apical membrane
of the distal tubules. The high alkaline phosphatase
activity in the PM fraction also supports this assumption.
Studies with marker enzymes also suggested differences between the BLM- and PM-enriched fractions.
The ratios of activities of Na+K+-ATPase to Mg2"1-ATPase differed 10-fold in the PM and BLM fractions; Na+K+-ATPase activity was concentrated in
the BLM fraction. This is in agreement with the findings of others, who localized this enzyme on the BLM
of the distal tubules by a histochemical technique.11-M
The plasma membrane marker enzyme alkaline
phosphatase activity was enriched 11-fold in the PM
TABLE 5. Activation of Renal Kallikrem in the BLM Fraction by Trypsin
Kallikrein activity
Source
Control
S-2266 hydrolysis
0.15 ± 0.03
(nmol/min/mg)
RIA of kinin released
0.69 ± 0.07
(ng/min/mg)
BLM + Triton
S-2266 hydrolysis
1.85 ±0.3
(nmol/min/mg)
X-100
RIA of kinin released
11.3 ±1.01
(ng/min/mg)
BLM = basolateral membrane; RIA = radioimmunoaasay.
Mean values ± 8EM. n = 5.
BLM
Activation by
Sepharosetrypsin
trypsin
0.47 ± 0.1
1.25 ±0.15
1.21 ±0.13
1.15 ±0.1
5.9 ± 0.9
7.8 ± 0.9
23.5 ± 1.31
24.2 ±1.2
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PROCEEDINGS/INTERAMERICAN SOCIETY
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fraction,14 but decreased to 0.45 in the BLM enriched
fraction over the crude homogenate.
The PM fraction, which was homogenized harsher
than the BLM, contained mostly empty vesicles.14 The
BLM fraction also contained vesicles of various sizes,
as shown by transmission electronmicroscopy, but
freeze fracture showed intact segments with infolding
of the membranes. Electron microscopic studies support our notion that a large percentage of our BLM
fraction originates from the infoldings of distal membrane in convoluted tubules. Our procedure seems to
preserve considerably larger membrane fragments and
vesicles than the method of Ebel et al., M which yielded
vesicles smaller than 1 /im.
The ultrastructure of the distal tubular cells indicates that the surface area of BLM is larger than the
PM on the luminal side. In secretory glands kallikrein is present in cells that are rich in mitochondria
and contain infoldings of cell membranes, a structure
usually associated with water and ion transport." The
basal part of the epithelium in distal tubules, which
faces the peritubular capillaries, also shows concentrated mitochondria in addition to infoldings of membranes." The isolated BLM fraction contained some
mitochondria with the membrane fragments enveloped around them even after many washings.
Hypothetically, there could be a functional relationship between kallikrein on BLM membrane and enzymes in the mitochondria.
Corthorn et al.u found a prekallikrein in human
urine that was activated by trypsin. Prekallikreins in
the rat kidney may be activated by serine proteases
that cleave peptide bonds at a basic amino acid residue
similar to trypsin; plasmin may be such an enzyme
(Yamada and Erd5s, unpublished). A location of kallikrein on the basal membrane suggests access to
plasma kininogen in the extracellular space. In addition, BLM kallikrein could be released in the renal
lymph and venous effluent. Kinins released in the
kidney may influence renal vascular resistance.
In conclusion, a significant amount of the total
kallikrein activity in the rat kidney is present as prekallikrcin and as a membrane-bound but inactive enzyme in the BLM fraction. This BLM kallikrein may
be the source of renal kallikrein that appears in renal
lymph and venous effluent of the perfused kidney,
while kallikrein and prekallikrein bound in the PMenriched fraction may be excreted in the urine.
Acknowledgments
We are grateful for the assistance of Anna Slier in electron
microscopy and for the advice of Dr. Y. Levin, of the Weizmann
Institute
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K Yamada, W W Schulz, D S Page and E G Erdös
Hypertension. 1981;3:II-59
doi: 10.1161/01.HYP.3.6_Pt_2.II-59
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