JW: On the mechanism of angiotensin-induced proteinuria. I. Studies in aminonucleoside nephrotic rats and in saralasin blockade. Nephron. 1981 ;24:47-50.
Angiotensin II Modulates Glomerular Capillary Permselectivity in Rat Isolated Perfused Kidney1 Radoslaw Giuseppe
Lapinski, Remuzzi
R. Lapinski,
N. Perico,
G. Remuzzi, Mario Research, Bergamo,
Norberto
A. Remuzzi, Negri Institute Italy
N. Perico, G. Remuzzi, Dialysis, Ospedali Riuniti (J. Am.
Soc.
Nephrol.
Perico,2 Andrea
Remuzzi,
F. Sangalli. A. Benigni, for Pharmacological
Division of di Bergamo,
Nephrology Bergamo,
and Italy
199#{243}; 7:653-#{243}#{243}O)
ABSTRACT Studies in experimental animals and humans have documented that inhibition of the renin-angiotensin system by angiotensin-converting enzyme inhibitors reduces urinary protein excretion rate and retards the development of renal injury. Here we sought to investigate whether angiotensin II (All) modified the sizeselective properties to macromolecules of the gbmerular capillary barrier in isolated perfused rat kidney preparation. Compared with basal values, continuous All infusion into the renal artery at the rate of 3 or 8 ng/min, but not at 0.6 ng/min, induced a
Fabio Sangalli,
sistently retards
the
Ariela
reduces urinary development
Benigni,
protein ofrenal
and
excretion injury. There
rate are
and also
some data both in experimental animals (3,9-1 1) and humans ( 1 2-1 5) that ACE inhibitors are superior to other antihypertensive drugs in retarding renal disease progression. The mechanism by which ACE inhibitors improve the selective properties of glomerular barrier is still a matter of intensive investigation. Besides the effect that ACE inhibitors have on lowering intraglomerular capillary pressure ( 1-3), that can by itself have a major impact on gbomerular permeability-as already documented in non-glomerular vascular beds ( 1 6)-there are reasons to believe that ACE inhibitors retard the ifitration of macromolecules by diredily interfering with glomerular barrier sizeselective function (4, 1 7-19). The fact that the effect of ACE inhibitors on glomerular size-selectivity is a result of their effect in blocking the generation of anglotensin nists improve
II (All) rests on data that All receptor antagothat are specific for Type 1 receptors also glomerular barrier permeability to macromol-
excretion rate. Evaluation of the sieving properties of the gbomerular barrier by fractional clearance of polydisperse Ficoll showed that All significantly enhanced the filtration oftracer molecules of radii 34A. All-induced changes in urinary protein excretion rate and in Ficoll fractional clearance were completely prevented by pretreatment with the specific All Type 1 receptor antagonist SR 47436.
ecules and retard the progression of the disease in experimental (20,2 1 ) and human (22) renal diseases. Here we sought to investigate whether All modified the size-selective properties of the glomerular barrier that is the organization and the distribution of membrane pores. To evaluate the effects that were directly linked to the action of All independent of systemic factors that may confound interpretation of the results, we used a preparation of isolated perfused kidney (23). Hemodynamic modifications have been minimized by maintaining a constant perfusion pressure in the preparation throughout the experiment.
Key Words: clearance,
METHODS
progressive
M
and
significant
Proteinuria, size-selective
increase
angiotensin properties
in urinary
receptor
protein
blockade,
ost
Rcoil
human renal diseases progress to endstage renal insufficiency even independently from the initial insult. This is particularly true for diseases that manifest with increased gbomerular permeabihity to macromolecules. Studies in experimental animals ( 1-4) and humans (5-8) have documented that inhibition of the renin-angiotensin system by angiotensin-converting enzyme (ACE) inhibitors con-
Adult male Sprague-Dawley rats (Charles River Italia Spa., Calco, Italy) were used in these studies. Animal care and treatment was conducted in conformity with institutional guidelines in compliance with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, December 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). All animals were allowed free access to standard rat chow and to tap water.
Experimental 1
Received
March
2 Correspondence
Research,
via
13, 1995.
Accepted
to Dr. N. Perico, Gavazzeni
1 1, 24125
December Mario Bergamo,
20, 1995.
Negri
Institute
Italy.
1046.6673/0705-0653$03.00/0 Journal of the American society of Nephrology Copyright C 1996 by the American Society of Nephrology
Journal
of the
American
Society
of Nephrology
for
Pharmacological
Design
To assess whether All directly affects glomerular permeability to macromolecules, a dose-response study was designed in an isolated perfused rat kidney preparation that is free of the influence of systemic factors (e.g. , circulating vasoactive hormones) and of fluctuations in systemic blood
653
Angiotensin
II and
Glomerular
Permselective
Function
pressure and extracellular volume (23). Rat kidneys were isolated and perfused in a recirculating system at a constant pressure of 100 mm Hg with an artificial cell-free medium. After a 20- to 25-min equilibration period, a lO-min baseline urine collection and a perfusate sample were obtained at the end of the clearance period. A continuous infusion into the renal artery ofAJI or vehicle (N = 6) was then started and five consecutive clearance periods ( 1 0 mm each) were performed. For each collection period, urine and venous effluent sampies were analyzed for creatinine concentration. Urinary protein concentration was also determined. Urine output, urinary protein excretion rate, GFR, and renal perfusate flow (RPF) were measured. For these experiments, three groups of isolated kidneys infused with All at the rate of 0.6 (N = 6), 3 (N = 6), and 8 (N = 6) ng/mln, respectively, were considered. Moreover, giomerular size-selective permeabifity properties by the fractional clearance of endogenous Ficoll were determined on the baseline (0 to 10 mm perfusion time) and the last experimental clearance (50 to 60 min perfusion time) samples in isolated kklneys infused with 8 ng/rnin An. To investigate whether the possible changes in permsehective properties induced by All occurred through the binding ofAll to its Type 1 receptor (24), additional experiments were performed in the presence of All receptor blockade. For this purpose, rats were given (5 mg/kg, N = 4) the new potent and selective 1’ype I All receptor antagonist SR 47436 (2-n-butyl3-E(2’-(1H-tetrazoh-5-yl)-biphenyl-4-yl)methyh]1 ,3-dlazaspiro[4,4lnon1 -en-4-one; Sanofi Recherche, Montpellier, France) (25) or its vehicle (N = 3) orally 2 h before initiating perfusion of the right kidney. The dose of SR 47436 was chosen on the basis of previous data in normotensive rats showing that this All receptor antagonist, when given orally at the dose of 0.3 to 30 mg/kg, prevented the AII-pressor response in a dose-related manner (25). After surgery, the equilibration period, and a baseline clearance period, All infusion at the rate of 8 ng/mln was started and five consecutive clearance periods were performed. GFR, RPF, urine output, urinary protein excretion rate, and fractional clearance of native Ficoll molecules were measured as above.
Isolated
Rat Kidney
Perfusion
The perfusion technique used in these experiments has been previously described in detail (26). In brief, the rats were anesthetized with thiopental sodium (50 mg/kg body wt ip) and placed on a heated surgical table. The abdominal cavity was exposed and the vena cava tributaries below the right renal pedicle and above the iliac bifurcation were tied. After iv injection of 0.5 mL of 10% mannltol and 120 U of heparin, the right ureter was isolated from the surrounding connective tissue and cannulated with PE- 10 polyethylene tubing (Clay-Adams, Parsippany, NJ). The open tip of a venous cannula (PE-240 polyethylene catheter) with its dEstal end closed was introduced into the vena cava below the right renal vein and secured in place. The renal artery was then cannulated with a short, blunted 19-gauge needle via the superior mesenteric artery to avoid interruption offlow to the kidney. The arterial cannula was secured in place by ligatures and the distal end of the venous cannula was opened. Ligatures around the vena cava above the right renal pedicle were tied. At this time, the rat was kified. After being washed with 50 mL of oxygenized perfusion solution, the kidney was perfused In situ in a recirculating system with a medium held at 37#{176}C by a constant Haake Dl temperature circulator (Haake, Berlin, Germany) and gassed with a mixture of 95% 02-5% CO2 through a hollow-fiber membrane
654
oxygenator. The perfusate was delivered to the renal artery cannula through a peristaltic pump (Gilson 2, Vilhier LeBel, France), an in-line 8-gm-pore-size ifiter (Sartorious, Gottingen, Germany), and a glass bubble trap. The perfusate consisted of Krebs-Henseleit bicarbonate buffer containing 3.5 g/dL of Ficoll 70 (Pharmacia Fine Chemicals, Uppsala, Sweden), 1 g/dL of BSA (Pentex BSA Fraction V, Miles Laboratories, Elkhart, IN), 200 mg/dL of glucose, 36 mg/dL of urea, 50 mg/dL of creatinine, and a mixture of amino acids that included methionine (0.5 mM), alanine (2.0 mM), glycine (2.0 mM), serine (2.0 mM), arginine (1 .0 mM), isoleucine (1 .0 mM), aspartic acid (3.0 mM), and cysteine (0.5 MM). The perfusate was ifitered through a 0.45-pm-pore-size membrane ifiter (Sartorius) before use and, when equifibrated with the gas mixture at 37#{176}C, its pH was approximately 7.4. The total volume of the perfusate in the system was 250 mL. Urine flow was determined gravimetrically. GFR was calculated as creatinine clearance. This has been shown to give the same GFR estimates in the isolated perfused kidney as inulin clearance (27). RPF was determined volumetrically. The perfusion pressure was continuously measured with a Statham transducer (Gould, Dusseldorf, Germany) connected by polyethylene tubing to the perfusion line proximally to the arterial cannula. The effective perfusion pressure (cannula tip pressure) was derived by subtracting from the measured pressure the pressure drop known to occur across the arterial cannula at a given flow, and was kept constant at 100 mm Hg throughout the experiments. Therefore, changes in RPF reflected changes in renal vascular resistance. Total urinary protein excretion rate was measured in duplicate samples by the Coomassie brffliant blue dye-binding assay (28).
Ficoll Clearance
Determination
Graded-size Ficoll molecules in perfusate and urine sampies were separated by gel-permeation chromatography on an Sephacril 5-300 HR column (1 .6 X 40 cm; Pharmacia) as previously described (2 1). Column calibration was performed with six Ficoll fractions of known molecular weight (range, 17,500 to 132,000) kIndly provided by Dr. K. Granath (Pharmacia). Effective molecular radII for Ficoll in eluted fractions were calculated according to Ficoll diffusion coefficients measured by Oliver et at. (29) using quasielastic light scattering. According to these observations, the relationship between weight-average molecular weight (Mj and effective molecular radius (r) is r
=
0.421
x (M)#{176}427
where r is given in A. In each experiment, the fractional clearance of Ficoll was measured in the first clearance period (0 to 10 mm perfusion time, basal) and in the last clearance (from 50 to 60 mm perfusion time). Protein-free samples of perfusate and urine fluid were separated, fractions of approximately 2 mL were automatically collected on the gel column, and Ficoll concentration was subsequently assayed in eluted fractions by the anthrone method with slight modifications (30,31). Fractional clearance of Ficoll was calcuhated as Op(U/P)/(U/P)r, where (U/P)F and (U/P)r are the urine-to-plasma concentration ratios of Ficoll and creatinine, respectively. Ficoll molecules with an effective molecular radius ranging from 18 to 50 A were considered.
Volume
7
.
Number
5
‘
1996
Laplnskl
Statisticab Anabysis All results are expressed as mean ± SD. Data were anahyzed using the t test for paired data or two-way analysis of variance as appropriate. The significance level of differences between individual group means, subjected to the analysis of variance, was established using the Tukey-Cicchetti test for multiple comparisons (32). The statistical significance level was defined as P < 0.05.
RESULTS Effect of All on Renal Functional and Gbomerular Size-Selectivity
values
not
significantly
from
different
ng/min
time ence
values.
Increasing
trast,
8 ng/min All sustained decrease significance 20 min
vasoactive As
the
resulted in a numerical as compared with baseline did not reach statistical
in kidneys
peptide
shown
infusion
rate
to
a progressive
2, no
significant
changes
quite
rate
constant
3
during
perfusion
and
All
effect
of angiotensin
II (All)
not
of Ficoll
change
intrarenal of All did not
dose
significantly
molecules increased
Table
5 shows
isolated
kidneys
receptor
experi-
infusion
affect
of
signifi-
larger after
All
the
sieving
coef-
(ranging from 1 8 to the fractional clearthan 34A in radius infusion.
All Type 1 Receptor
the
from
renal
functional
rats
parameters
pretreated
with
in
the
All
SR 47436 and exposed to 8 ng/ SR 47436 pretreatment did not affect urine output, which also remained quite constant during All infusion. Exposure of isolated kidneys to the All receptor antagonist completely prevented the decline in RPF and GFR induced by All. As shown in Figure 3, in the presence ofSR 47436, urinary protein excretion rate did not increase after kidneys were perfused with All. Thus, in these isolated kidneys,
min
All
protein
at
rable
or 3 ng/rnin
1 . Dose-response
did
Effect of a Specific
in GFR
with
in all of the
the
Antagonist
or with vehicle. When 8 ng/min All was infused, FF increased to a significant extent as compared with baseline values. Figure 1 reports the dose-response effect of All on the urinary protein excretion rate. Before perfusion of the kidneys with All or vehicle, the basal urinary TABLE
of 0.6
lowest
significantly
observed over the 50 mm of perfusion when All (0.6 or 3 ng/min) or vehicle were added to the perfusate with respect to preinfusion values. At the rate of 8 ng/min, All GFR was numerically but not significantly reduced. Table 3 shows ifitration fraction (FF) values. FF the
All
ance
were
remained
similar
protein
(29).
reached statistical after exposure of the kidney to the as compared with preinfusion values.
in Table
or the
was
ficients of small Ficoll molecules 34,A in radius). On the contrary,
decrease in RPF with values, but the differsignificance. By con-
infusion caused in flow that
vehicle
rate Although
excretion rate during the entire obserperiod, All at the rate of 3 ng/min induced a mild but progressive increase in urinary protein excretion rate. The effect of All was more prominent when the infusion rate was raised to 8 ng/min. The increase in urinary protein excretion rate was observed within 10 min after kidney exposure to All, and 10 mm hater reached statistically significant values as compared with preinfusion ones. Fractional clearance values for Ficoll molecules of graded sizes before and after All infusion are reported in Table 4 and in Figure 2. The Ficohl sieving coefficient for smaller molecules ( 18 A in radius) during basal evaluation approached 0.5, in line with corresponding values reported for the intact normal kidney in the rat
exposed to vehicle alone (from 90 to 1 12 L/min). The urine output of isolated kidneys infused with 8 ng/ min was also in the same range (from 98 to 125 L/min) despite values that were numerically higher. Table 1 reports the dose-response effect of All on RPF. All infusion at the rate of 0.6 ng/min did not change RPF to a significant extent with respect to preinfusion
groups.
cantly
function in a exposed to 0.6 ranged from 95 respectivelythose
excretion
mental
vation
Parameters
The All infusion modulated renal dose-dependent manner. In kidneys and 3 ng/mln All, the urine flow rate to 1 15 and from 92 to 120 p.L/mln,
protein
et al
antagonist
infusion.
excretion with
infusion,
rate during
preinfusion 6 ± 2 p.g/min).
All
values
infusion
was
(basal,
In line
with
4.5 this
compa1 .3; All
±
observation,
fractional clearances of small and large Ficoll molecules in kidenys pretreated with SR 47436 and infused with All were similar to preinfusion values (see Table 4 and Figure 4).
on
renal
perfusate
flow
In isolated
perfused
rat kidneysa
RPF (mi/mm) Time
(mm)
Vehicle All(0.#{243}ng/min) All(3ng/mmn) All(8ng/mln) 0 b C
10b
20
30
40
50
60
27±3 26±2 27±4 27±3
27±3 25±3 25±3
28±3 24±3 25±4
27±3 24±2 23±2
28±3
28±3
24±2
24±2
23±2
23±2
19±3
17±4c
17±4c
17±4c
17±4c
Values are mean ± SD. Baseline prelnfuslon values. p < 0.01 versus basal values.
Journal
of the
American
Society
of Nephrology
655
Angiotensin
II and
Glomerular
Permselective
TABLE 2. Dose-response
effect
Function
of All on GFR in isolated
perfused
rat kidneysa per g kidney
GFR (mi/mm Time (mm)
20
10b
a b
Vehicle
0.508
±
All (0.6 ng/mmn) All (3 ng/mmn) All (8 ng/mmn)
0.627 0.561 0.634
±
Values are mean Baseline preinfusion
0.065 0.102 0.133 0.107
± ±
30
0.534
±
0.599
±
0.076 0.084
0.543 0.578
±
0.510 0.462
± ±
0.094
0.461
±
0.126
0.437
±
±
wt) 50
40
0.085 0.105 0.071 0.129
0.546 0.570 0.442
±
0.542 0.557 0.424
±
±
0.059 0.076 0.089
0.440
±
0.125
0.414
±
±
± ±
60 0.068 0.076 0.084 0.136
0.529 0.520 0.427 0.405
± ± ±
±
0.059 0.090 0.105 0.120
± SD.
values.
TABLE 3. Dose-response
effect
of All on filtration
fraction
(FF) in isolated
perfused
rat kidneysa
FF Time
(mm)
a b C
30
20
10b
Vehicle All (0.6 ng/mmn) All (3 ng/min)
0.019 0.024 0.020
±
0.002 0.002
0.019
±
±
0.024
±
All (8 ng/min)
0.021
±
± 0.004
0.007
0.023
±
0.024
±
0.003 0.002 0.005 0.005
0.020 0.023 0.022 0.028
± ± ± ±
40 0.003 0.002 0.003 0.002c
0.020 0.024 0.022 0.029
± ± ± ±
50
60
0.002 0.002 0.003 0.003c
0.020 0.023 0.022 0.029
0.019 0.022 0.021
±
±
0.002 0.001 0.005
±
0.004c
0.029
±
findings
that
pressure
± ±
± ±
0.002 0.003 0.006 0.005c
Values are mean ± SD. Baseline preinfusion values. p < 0.05 versus basal values.
umented
by
60
excretion FF
40
0’-
0
10
20
30
Time
(pre-All,
at
40
50
60
(minutes)
1 . Dose-response effect of All infusion on urinary excretion rate in Isolated perfused rat kidney prepaValues are mean ± SD. P < 0.05 versus basal
10 mm);
*
basal
versus
34A were
configuration
clearance significantly
macromolecules by also
All infusion. indicate that
size-selectivity
similar
of Ficoll increased
smaller
than
The data All-induced
of
of alnot
presented increase
is a function
Volume
that
molecules by All, 34A were
to
in in
this gb-
of intracellular
7
‘
Number
5
-
1996
Lapinski
TABLE 4. Fractional SR47436 + All
clearance
values
in basal
condition
and
during
infusion
Basal
All
Basal
with All or during
SR47436
(N =8) 18 20 22
0.410 0.396
±
0.378
±
24
±
26 28
0.345 0.299 0.255
30
0.207
±
32 34 36
0.162
0.427 0.409
±
0.152 0.129
0.384
±
0.354
±
0.319
±
±
0.095 0.072 0.052 0.038
0.283 0.248 0.210
0.121
±
0.028
0.179
0.090
±
38
0.066
±
40
0.047
±
42
0.033
±
44
0.024 0.016 0.012 0.009
±
0.024 0.021 0.016 0.013 0.010 0.009 0.007 0.006
48 50
C
(N=
0.176 0.154
46
b
Values are mean ± SD. p < 0.05 versus Basal. p < ow versus Basal.
signals
delivered
SP47436.
through
±
± ±
± ± ±
receptor
All
All
Type
antagonist;
0.327 0.314
0.467 0.460
±
0.431 0.394 0.339 0.281
±
±
0.278 0.232 0.188 0.149
±
0.1 12
0.226
±
0.078
0.166
±
0054b
0.153 0.129 0.1 1 1 0.096 0.082
±
0.072
±
0.064 0.067
±
0.045c 0.044c 0.049c 0.050 0.050w 0.049c 0.046c 0.039c
±
± ± ± ±
±
lated
perfused
ance
1 receptors,
All
kidney,
of barge
but
not
enhancing
small
the
dextran
actin
fractional
clear-
macromolecules
polymerization
(40).
a)
and
the
size-
and
charge-
selective function of the barrier ( 19) is that although the albumin excretion rate increased more than eight times upon infusion of All (from 8.0 ± 4.2 to 7 1 . 1 ± 22.6 g/min), the clearance of Ficohb molecules corresponding to the size of albumin (36A) (obtained by multiplying 0 for Ficoll molecules of 36A in radius by
American
± ±
±
0.300 0.227
±
0.068
0.166
±
0.113 0.070 0.044 0.029
0.1 15 0.078 0.053 0.035
±
0.048
0.1 17
±
0.022
±
0.033 0.022
0.080 0.055
±
0.023
±
0.015 0.010 0.006 0.005
± ±
0.014 0.009 0.006 0.004
±
0.003
±
0.002
0.037 0.025 0.017 0.012 0.009 0.006
0.015 0.010 0.007 0.005 0.003 0.003 0.003 0.002
Society
of Nephrology
±
± ± ±
±
± ± ± ± ± ± ±
0.327 0.260
0.229 0.168
All ,,,..
Basal
0.200
0.100
-
Ca C
0 C.) Ca U-
S
0.050
0.020
0.010
cell
hemodynamics
of the
0.387
0.1 14 0.093
±
C.) C
Because
foot processes have a highly suggestive of contractile funcexists that All may alter the permsebective properties of the gbomerubar barrier by mediating contraction within the foot process and enhancing the size of gbomerubar slit pores. An additional finding of our study that may contribute to the clarification of the relationship between gbomeruhar
0.125
±
...,,.
a)
epithelial cytoskeleton possibility
±
±
0.637 0.603 0.549 0.475
-0--
0
All-induced
±
0.154 0.155 0.138 0.126
±
1.000
The mechanisms underlying the effect of All on gbomerular sieving properties have to be explored further. By imaging glomeruli with confocal microscopy to visualize the thin optical cross-sections of glomeruhar capifiaries, it has recently been shown that the perfusion of rat kidney with All increased the quantity of F-actin in the ifitration barrier, consistent gbomerular developed tion, the
5)
as
(39).
Journal
+
P. radius.
documented by the finding that the selective All Type I receptor antagonist prevented the phenomenon. Thus All modulates gbomerular size-selectivity. We have previously shown that another hormone similar to All, platelet-activating factor (PAF), also increases glomeruhar permeability to macromolecules in iso-
with
with
(A)
R
a
infusion
et al
a
p