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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