Journal of the American Society of Nephrology 653

Angiotensin II Modulates Glomerular CapillaryPermselectivity in Rat Isolated Perfused Kidney1Radoslaw Lapinski, Norberto Perico,2 Andrea Remuzzi, Fabio Sangalli, Ariela Benigni, andGiuseppe Remuzzi

R. Lapinski, N. Perico, A. Remuzzi, F. Sangalli. A. Benigni,G. Remuzzi, Mario Negri Institute for PharmacologicalResearch, Bergamo, Italy

N. Perico, G. Remuzzi, Division of Nephrology andDialysis, Ospedali Riuniti di Bergamo, Bergamo, Italy

(J. Am. Soc. Nephrol. 199#{243};7:653-#{243}#{243}O)

ABSTRACTStudies in experimental animals and humans havedocumented that inhibition of the renin-angiotensinsystem by angiotensin-converting enzyme inhibitorsreduces urinary protein excretion rate and retards thedevelopment of renal injury. Here we sought to inves-tigate whether angiotensin II (All) modified the size-selective properties to macromolecules of the gb-merular capillary barrier in isolated perfused ratkidney preparation. Compared with basal values,continuous All infusion into the renal artery at the rateof 3 or 8 ng/min, but not at 0.6 ng/min, induced aprogressive and significant increase in urinary protein

excretion rate. Evaluation of the sieving properties ofthe gbomerular barrier by fractional clearance ofpolydisperse Ficoll showed that All significantly en-hanced the filtration oftracer molecules of radii �34A.All-induced changes in urinary protein excretion rateand in Ficoll fractional clearance were completelyprevented by pretreatment with the specific All Type 1receptor antagonist SR 47436.

Key Words: Proteinuria, angiotensin receptor blockade, Rcoil

clearance, size-selective properties

M ost human renal diseases progress to end-

stage renal insufficiency even independently

from the initial insult. This is particularly true for

diseases that manifest with increased gbomerular per-

meabihity 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-

1 Received March 13, 1995. Accepted December 20, 1995.

2 Correspondence to Dr. N. Perico, Mario Negri Institute for Pharmacological

Research, via Gavazzeni 1 1, 24125 Bergamo, Italy.

1046.6673/0705-0653$03.00/0Journal of the American society of NephrologyCopyright C 1996 by the American Society of Nephrology

sistently reduces urinary protein excretion rate and

retards the development ofrenal injury. There are 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 dis-

ease progression. The mechanism by which ACE in-

hibitors improve the selective properties of glomerular

barrier is still a matter of intensive investigation.

Besides the effect that ACE inhibitors have on lower-

ing intraglomerular capillary pressure ( 1-3), that can

by itself have a major impact on gbomerular perme-

ability-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 size-

selective function (4, 1 7-19). The fact that the effect of

ACE inhibitors on glomerular size-selectivity is a re-

sult of their effect in blocking the generation of anglo-

tensin II (All) rests on data that All receptor antago-nists that are specific for Type 1 receptors also

improve glomerular barrier permeability to macromol-

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 mem-

brane pores. To evaluate the effects that were directly

linked to the action of All independent of systemic

factors that may confound interpretation of the re-

sults, we used a preparation of isolated perfused

kidney (23). Hemodynamic modifications have been

minimized by maintaining a constant perfusion pres-

sure in the preparation throughout the experiment.

METHODSAdult male Sprague-Dawley rats (Charles River Italia

Spa., Calco, Italy) were used in these studies. Animal careand treatment was conducted in conformity with institu-tional guidelines in compliance with national and interna-tional laws and policies (EEC Council Directive 86/609, OJ L358, December 1987; NIH Guide for the Care and Use ofLaboratory Animals, NIH Publication No. 85-23, 1985). Allanimals were allowed free access to standard rat chow and totap water.

Experimental DesignTo assess whether All directly affects glomerular perme-

ability to macromolecules, a dose-response study was de-signed in an isolated perfused rat kidney preparation that isfree of the influence of systemic factors (e.g. , circulatingvasoactive hormones) and of fluctuations in systemic blood

Angiotensin II and Glomerular Permselective Function

654 Volume 7 . Number 5 ‘ 1996

pressure and extracellular volume (23). Rat kidneys wereisolated and perfused in a recirculating system at a constantpressure of 100 mm Hg with an artificial cell-free medium.After a 20- to 25-min equilibration period, a lO-min baselineurine collection and a perfusate sample were obtained at theend of the clearance period. A continuous infusion into therenal artery ofAJI or vehicle (N = 6) was then started and fiveconsecutive clearance periods ( 1 0 mm each) were performed.For each collection period, urine and venous effluent sam-pies were analyzed for creatinine concentration. Urinaryprotein concentration was also determined. Urine output,urinary protein excretion rate, GFR, and renal perfusate flow(RPF) were measured. For these experiments, three groups ofisolated 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 propertiesby the fractional clearance of endogenous Ficoll were deter-mined on the baseline (0 to 10 mm perfusion time) and thelast 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 permsehec-tive properties induced by All occurred through the bindingofAll to its Type 1 receptor (24), additional experiments wereperformed in the presence of All receptor blockade. For thispurpose, rats were given (5 mg/kg, N = 4) the new potent andselective 1’ype I All receptor antagonist SR 47436 (2-n-butyl-3-E(2’-(1H-tetrazoh-5-yl)-biphenyl-4-yl)methyh]- 1 ,3-dlaza-spiro[4,4lnon- 1 -en-4-one; Sanofi Recherche, Montpellier,France) (25) or its vehicle (N = 3) orally 2 h before initiatingperfusion of the right kidney. The dose of SR 47436 waschosen on the basis of previous data in normotensive ratsshowing that this All receptor antagonist, when given orallyat the dose of 0.3 to 30 mg/kg, prevented the AII-pressorresponse in a dose-related manner (25). After surgery, theequilibration period, and a baseline clearance period, Allinfusion at the rate of 8 ng/mln was started and five consec-utive clearance periods were performed. GFR, RPF, urineoutput, urinary protein excretion rate, and fractional clear-ance of native Ficoll molecules were measured as above.

Isolated Rat Kidney Perfusion

The perfusion technique used in these experiments hasbeen previously described in detail (26). In brief, the ratswere anesthetized with thiopental sodium (50 mg/kg body wtip) and placed on a heated surgical table. The abdominalcavity was exposed and the vena cava tributaries below theright renal pedicle and above the iliac bifurcation were tied.After iv injection of 0.5 mL of 10% mannltol and 120 U ofheparin, the right ureter was isolated from the surroundingconnective tissue and cannulated with PE- 10 polyethylenetubing (Clay-Adams, Parsippany, NJ). The open tip of avenous cannula (PE-240 polyethylene catheter) with its dEs-tal end closed was introduced into the vena cava below theright renal vein and secured in place. The renal artery wasthen cannulated with a short, blunted 19-gauge needle viathe superior mesenteric artery to avoid interruption offlow tothe kidney. The arterial cannula was secured in place byligatures and the distal end of the venous cannula wasopened. Ligatures around the vena cava above the right renalpedicle were tied. At this time, the rat was kified. After beingwashed with 50 mL of oxygenized perfusion solution, thekidney was perfused In situ in a recirculating system with amedium held at 37#{176}Cby a constant Haake Dl temperaturecirculator (Haake, Berlin, Germany) and gassed with a mix-ture of 95% 02-5% CO2 through a hollow-fiber membrane

oxygenator. The perfusate was delivered to the renal arterycannula through a peristaltic pump (Gilson 2, Vilhier LeBel,France), an in-line 8-gm-pore-size ifiter (Sartorious, Gottin-gen, Germany), and a glass bubble trap.

The perfusate consisted of Krebs-Henseleit bicarbonatebuffer containing 3.5 g/dL of Ficoll 70 (Pharmacia FineChemicals, Uppsala, Sweden), 1 g/dL of BSA (Pentex BSAFraction V, Miles Laboratories, Elkhart, IN), 200 mg/dL ofglucose, 36 mg/dL of urea, 50 mg/dL of creatinine, and amixture 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), andcysteine (0.5 MM). The perfusate was ifitered through a0.45-pm-pore-size membrane ifiter (Sartorius) before useand, when equifibrated with the gas mixture at 37#{176}C,its pHwas approximately 7.4. The total volume of the perfusate inthe system was 250 mL.

Urine flow was determined gravimetrically. GFR was cal-culated as creatinine clearance. This has been shown to givethe same GFR estimates in the isolated perfused kidney asinulin clearance (27). RPF was determined volumetrically.The perfusion pressure was continuously measured with aStatham transducer (Gould, Dusseldorf, Germany) con-nected by polyethylene tubing to the perfusion line proxi-mally to the arterial cannula. The effective perfusion pres-sure (cannula tip pressure) was derived by subtracting fromthe measured pressure the pressure drop known to occuracross the arterial cannula at a given flow, and was keptconstant at 100 mm Hg throughout the experiments. There-fore, changes in RPF reflected changes in renal vascularresistance. Total urinary protein excretion rate was mea-sured in duplicate samples by the Coomassie brffliant bluedye-binding assay (28).

Ficoll Clearance Determination

Graded-size Ficoll molecules in perfusate and urine sam-pies were separated by gel-permeation chromatography onan Sephacril 5-300 HR column (1 .6 X 40 cm; Pharmacia) aspreviously described (2 1). Column calibration was performedwith six Ficoll fractions of known molecular weight (range,17,500 to 132,000) kIndly provided by Dr. K. Granath (Phar-macia). Effective molecular radII for Ficoll in eluted fractionswere calculated according to Ficoll diffusion coefficients mea-sured by Oliver et at. (29) using quasielastic light scattering.According to these observations, the relationship betweenweight-average molecular weight (M�j and effective molecu-lar radius (r) is

r = 0.421 x (M�)#{176}427

where r is given in A. In each experiment, the fractionalclearance 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 ofperfusate and urine fluid were separated, fractions of ap-proximately 2 mL were automatically collected on the gelcolumn, and Ficoll concentration was subsequently assayedin eluted fractions by the anthrone method with slight mod-ifications (30,31). Fractional clearance of Ficoll was calcu-hated as Op�(U/P)�/(U/P)�r, where (U/P)F and (U/P)�r are theurine-to-plasma concentration ratios of Ficoll and creati-nine, respectively. Ficoll molecules with an effective molecu-lar radius ranging from 18 to 50 A were considered.

Laplnskl et al

Journal of the American Society of Nephrology 655

Statisticab AnabysisAll results are expressed as mean ± SD. Data were ana-

hyzed using the t test for paired data or two-way analysis ofvariance as appropriate. The significance level of differencesbetween individual group means, subjected to the analysis ofvariance, was established using the Tukey-Cicchetti test formultiple comparisons (32). The statistical significance levelwas defined as P < 0.05.

RESULTSEffect of All on Renal Functional Parametersand Gbomerular Size-Selectivity

The All infusion modulated renal function in adose-dependent manner. In kidneys exposed to 0.6and 3 ng/mln All, the urine flow rate ranged from 95to 1 15 and from 92 to 120 p.L/mln, respectively-values not significantly different from those in kidneys

exposed to vehicle alone (from 90 to 1 12 �L/min). Theurine 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 onRPF. All infusion at the rate of 0.6 ng/min did notchange RPF to a significant extent with respect topreinfusion values. Increasing the infusion rate to 3

ng/min resulted in a numerical decrease in RPF withtime as compared with baseline values, but the differ-ence did not reach statistical significance. By con-trast, 8 ng/min All infusion caused a progressive and

sustained decrease in flow that reached statisticalsignificance 20 min after exposure of the kidney to thevasoactive peptide as compared with preinfusion values.

As shown in Table 2, no significant changes in GFR

were observed over the 50 mm of perfusion when All

(0.6 or 3 ng/min) or vehicle were added to the perfus-ate with respect to preinfusion values. At the rate of 8ng/min, All GFR was numerically but not significantlyreduced.

Table 3 shows ifitration fraction (FF) values. FFremained quite constant during perfusion with All atthe rate of 0.6 or 3 ng/rnin or with vehicle. When 8ng/min All was infused, FF increased to a significantextent as compared with baseline values.

Figure 1 reports the dose-response effect of All onthe urinary protein excretion rate. Before perfusion ofthe kidneys with All or vehicle, the basal urinary

protein excretion rate was similar in all of the experi-mental groups. Although the intrarenal infusion of

vehicle or the lowest dose of All did not affect signifi-cantly protein excretion rate during the entire obser-vation period, All at the rate of 3 ng/min induced amild but progressive increase in urinary protein ex-cretion rate. The effect of All was more prominentwhen the infusion rate was raised to 8 ng/min. Theincrease in urinary protein excretion rate was ob-served within 10 min after kidney exposure to All, and10 mm hater reached statistically significant values ascompared with preinfusion ones.

Fractional clearance values for Ficoll molecules ofgraded sizes before and after All infusion are reported

in Table 4 and in Figure 2. The Ficohl sieving coefficientfor smaller molecules ( 18 A in radius) during basalevaluation approached 0.5, in line with correspondingvalues reported for the intact normal kidney in the rat(29). All did not change significantly the sieving coef-

ficients of small Ficoll molecules (ranging from 1 8 to34,A in radius). On the contrary, the fractional clear-ance of Ficoll molecules larger than 34A in radiussignificantly increased after All infusion.

Effect of a Specific All Type 1 ReceptorAntagonist

Table 5 shows the renal functional parameters inisolated kidneys from rats pretreated with the All

receptor antagonist SR 47436 and exposed to 8 ng/min All infusion. SR 47436 pretreatment did not affecturine output, which also remained quite constantduring All infusion. Exposure of isolated kidneys tothe All receptor antagonist completely prevented thedecline in RPF and GFR induced by All. As shown in

Figure 3, in the presence ofSR 47436, urinary proteinexcretion rate did not increase after kidneys wereperfused with All. Thus, in these isolated kidneys,protein excretion rate during All infusion was compa-

rable with preinfusion values (basal, 4.5 ± 1 .3; All

infusion, 6 ± 2 p.g/min). In line with this observation,

fractional clearances of small and large Ficoll mole-cules in kidenys pretreated with SR 47436 and in-fused with All were similar to preinfusion values (seeTable 4 and Figure 4).

TABLE 1 . Dose-response effect of angiotensin II (All) on renal perfusate flow In isolated perfused rat kidneysa

Time (mm)RPF (mi/mm)

10b 20 30 40 50 60

Vehicle 27±3 27±3 28±3 27±3 28±3 28±3All(0.#{243}ng/min)All(3ng/mmn)All(8ng/mln)

26±227±427±3

25±325±319±3

24±325±417±4c

24±223±217±4c

24±223±217±4c

24±223±217±4c

0 Values are mean ± SD.b Baseline prelnfuslon values.C p < 0.01 versus basal values.

60

40

20

0’-

Angiotensin II and Glomerular Permselective Function

656 Volume 7 ‘ Number 5 - 1996

TABLE 2. Dose-response effect of All on GFR in isolated perfused rat kidneysa

GFR (mi/mmTime (mm)

10b 20 30

per g kidney wt)

40 50 60

Vehicle 0.508 ± 0.065 0.534 ± 0.076 0.543 ± 0.085 0.546 ± 0.059 0.542 ± 0.068 0.529 ± 0.059All (0.6 ng/mmn) 0.627 ± 0.102 0.599 ± 0.084 0.578 ± 0.105 0.570 ± 0.076 0.557 ± 0.076 0.520 ± 0.090All (3 ng/mmn) 0.561 ± 0.133 0.510 ± 0.094 0.461 ± 0.071 0.442 ± 0.089 0.424 ± 0.084 0.427 ± 0.105All (8 ng/mmn) 0.634 ± 0.107 0.462 ± 0.126 0.437 ± 0.129 0.440 ± 0.125 0.414 ± 0.136 0.405 ± 0.120

a Values are mean ± SD.b Baseline preinfusion values.

TABLE 3. Dose-response effect of All on filtration fraction (FF) in isolated perfused rat kidneysa

Time (mm)10b 20 30

FF

40 50 60

Vehicle 0.019 ± 0.002 0.019 ± 0.003 0.020 ± 0.003 0.020 ± 0.002 0.020 ± 0.002 0.019 ± 0.002All (0.6 ng/mmn) 0.024 ± 0.002 0.024 ± 0.002 0.023 ± 0.002 0.024 ± 0.002 0.023 ± 0.001 0.022 ± 0.003All (3 ng/min) 0.020 ± 0.007 0.023 ± 0.005 0.022 ± 0.003 0.022 ± 0.003 0.022 ± 0.005 0.021 ± 0.006All (8 ng/min) 0.021 ± 0.004 0.024 ± 0.005 0.028 ± 0.002c 0.029 ± 0.003c 0.029 ± 0.004c 0.029 ± 0.005c

a Values are mean ± SD.b Baseline preinfusion values.C p < 0.05 versus basal values.

0 10 20 30 40 50 60

Time (minutes)

Figure 1 . Dose-response effect of All infusion on urinaryprotein excretion rate in Isolated perfused rat kidney prepa-rations. Values are mean ± SD. � P < 0.05 versus basal(pre-All, 10 mm); * � < � versus basal (pre-All, 10 mm).

DISCUSSIONWe have found that All caused a dose-dependent

increase in glomerular permeability to proteins in the

isolated perfused rat kidney preparation. This phe-

nomenon can be completely prevented by pretreatinganimals with a specific Type 1 All receptor antagonist.

These findings are consistent with studies tn vtvo in

the rat, in which renin or All enhanced the ifitration ofmacromolecules (33-36). This could well be a conse-

quence of increased intraglomerular pressure as doc-

umented by findings that pressure elevation in the

capillary human forearm may cause stretching of

microvascular membrane pores, which would in-

crease in diameter ( 1 6). Actually, in our preparation,

an increase in FF was found when All was infused at

the highest rate utilized in the study. On the other

hand, lowering the All infusion rate to 3 ng/min still

resulted in a progressive increase in urinary protein

excretion rate, despite no changes in FF. An increasedFF at 8 ng/min All infusion is indeed consistent with

an increased glomerular capillary pressure. Therefore,the above results can be taken to suggest that accord-

ing to the concentration of the hormone, All modu-

lates glomerular membrane permeability properties in

a complex way that implies hemodynamic and nonhe-

modynamic effects.

Epithelial cell slit diaphragms were considered toplay an important role in the sieving barrier of glomer-ular capifiaries (19,37). Glomerular basement mem-brane-a regular meshwork of collagen Type IV ar-

ranged with entactin, laminin, and heparan sulfate

molecules-is also believed, indeed, to impose a cer-tain sieving action against the passage of macromole-

cules across the glomerular capillaries (19,38).

In the study presented here, the effect of All onglomerular sieving properties has been explored bythe fractional clearance of polydisperse Ficoll, charac-terized by a globular configuration similar to that of

albumin. Fractional clearance of Ficoll molecules ofradii >34A were significantly increased by All, al-though macromolecules smaller than 34A were notaffected by All infusion. The data presented in this

study also indicate that All-induced increase in gb-

merular size-selectivity is a function of intracellular

1.000

-0-- All

...,,. ,,,.. Basal

0.200

0.100 -

a)C.)C

a)0CaC

0

C.)Ca

U-

S

0.050

0.020

0.010a p < 0.05 Vs. Basal

0.005**P<0,01 Vs. Basal

, I I

20 30 40 50

Lapinski et al

Journal of the American Society of Nephrology 657

TABLE 4. Fractional clearance values in basal condition and during infusion with All or during infusion withSR47436 + All�

R (A)Basal All Basal SR47436 + All

=8)(N (N= 5)

18 0.410 ± 0.176 0.427 ± 0.327 0.467 ± 0.154 0.637 ± 0.32720 0.396 ± 0.154 0.409 ± 0.314 0.460 ± 0.155 0.603 ± 0.260

22 0.378 ± 0.152 0.384 ± 0.278 0.431 ± 0.138 0.549 ± 0.22924 0.345 ± 0.129 0.354 ± 0.232 0.394 ± 0.126 0.475 ± 0.16826 0.299 ± 0.095 0.319 ± 0.188 0.339 ± 0.125 0.387 ± 0.113

28 0.255 ± 0.072 0.283 ± 0.149 0.281 ± 0.1 14 0.300 ± 0.070

30 0.207 ± 0.052 0.248 ± 0.1 12 0.226 ± 0.093 0.227 ± 0.044

32 0.162 ± 0.038 0.210 ± 0.078 0.166 ± 0.068 0.166 ± 0.02934 0.121 ± 0.028 0.179 ± 0054b 0.1 15 ± 0.048 0.1 17 ± 0.02236 0.090 ± 0.024 0.153 ± 0.045c 0.078 ± 0.033 0.080 ± 0.015

38 0.066 ± 0.021 0.129 ± 0.044c 0.053 ± 0.022 0.055 ± 0.010

40 0.047 ± 0.016 0.1 1 1 ± 0.049c 0.035 ± 0.014 0.037 ± 0.007

42 0.033 ± 0.013 0.096 ± 0.050� 0.023 ± 0.009 0.025 ± 0.005

44 0.024 ± 0.010 0.082 ± 0.050w 0.015 ± 0.006 0.017 ± 0.003

46 0.016 ± 0.009 0.072 ± 0.049c 0.010 ± 0.004 0.012 ± 0.003

48 0.012 ± 0.007 0.064 ± 0.046c 0.006 ± 0.003 0.009 ± 0.003

50 0.009 ± 0.006 0.067 ± 0.039c 0.005 ± 0.002 0.006 ± 0.002

a Values are mean ± SD. SP47436. All receptor antagonist; P. radius.b p < 0.05 versus Basal.C p < ow versus Basal.

signals delivered through All Type 1 receptors, as

documented by the finding that the selective All Type

I receptor antagonist prevented the phenomenon.Thus All modulates gbomerular size-selectivity. Wehave previously shown that another hormone similarto All, platelet-activating factor (PAF), also increasesglomeruhar permeability to macromolecules in iso-lated perfused kidney, enhancing the fractional clear-

ance of barge but not small dextran macromolecules(39).

The mechanisms underlying the effect of All ongbomerular sieving properties have to be exploredfurther. By imaging glomeruli with confocal micros-copy to visualize the thin optical cross-sections ofglomeruhar capifiaries, it has recently been shown thatthe perfusion of rat kidney with All increased thequantity of F-actin in the ifitration barrier, consistentwith All-induced actin polymerization (40). Because

gbomerular epithelial cell foot processes have a highlydeveloped cytoskeleton suggestive of contractile func-tion, the possibility exists that All may alter thepermsebective properties of the gbomerubar barrier bymediating contraction within the foot process andenhancing the size of gbomerubar slit pores.

An additional finding of our study that may contrib-ute to the clarification of the relationship betweengbomeruhar hemodynamics and the size- and charge-

selective function of the barrier ( 19) is that althoughthe albumin excretion rate increased more than eighttimes upon infusion of All (from 8.0 ± 4.2 to 7 1 . 1 ±

22.6 �g/min), the clearance of Ficohb molecules corre-sponding to the size of albumin (36A) (obtained bymultiplying 0 for Ficoll molecules of 36A in radius by

Effective Molecular Radius (A)

Figure 2. Fractional clearance of Ficoll of graded size distri-bution determined on the baseline (0 to 10 mm perfusiontime; Basal) and the last experimental clearance (50 to 60mm perfusion time; All) samples during All infusion (8 ng/mm) In isolated perfused rat kidney. � � < 0.05 versus basal;** P < 0.01 versus basal.

100�

80

600 Vehicle

+

. 0SR47436

*

*

*40

20

0

-0-- All#{247}SR47436

� Basal

0.200

0.100

. . - I I I I

10 20 30 40 50 60

0.050

Time (minutes)

0.020

0.010

0.005

I I I I I I I I I I I��1 I I I I f�[20 30 40 50

Anglotensin II and Glomerular Permselective Function

658 Volume 7 . Number 5 - 1996

TABLE 5. Effect of All receptor antagonist on changes in renal function induced by All lnfuslona

Time (mm)Vehicle SR 47436

Basalb Post-All Basalb Post-All

Diuresis (�L/min)

RPF (mi/mm)GFR (mi/mm per g kidney WI)

98 ± 12

28 ± 40.584 ± 0.105

105 ± 17

16 ± 3�0.423 ± 0.120

100 ± 15

27 ± 30.602 ± 0.1 15

108 ± 21

27 ± 40.528 ± 0.095

a Values are mean ± SD.b BaselIne prelnfuslon values.C p < o.oi versus basal values.

3C

.=

C.�54.-

0

Figure 3. Effect of pretreatment with the All receptor antago-nist SR 47436 on All-Induced protelnuria in isolated perfusedrat kidney preparations. Values are mean ± SD. � � < 0.01versus basal (pre-All, 10 mm).

a)C.)C

a)

0CaC0.5CS

U-

8U-

GFR) increased only by 18% on average (from 0.092 ±0.031 mL/min to 0. 108 ± 0.043 mL/min). These

results indicate that the changes in size-selectivityinduced by All can not entirely justify the massiveincrease in urinary protein excretion rate and suggestthat, in addition to a size defect, All may also consid-crabby impair the charge-selective function of the gb-merular barrier. The same issue has been raised by arecent study of Oliver et at. (4 1), who documented thatincreased albuminuria in nephrectomized fawn-hooded rats could not be completely explained by asize-selectivity defect but must be attributed to a

concomitant specific impairment of charge selectivity.It is tempting to speculate that permselective dysfunc-tion of the gbomerular capifiary wall as induced by All

may derive from development of shunt pathways inthe glomerular membrane that might be characterizedby loss of size- and charge-selectivity. One cannotexclude, however, that the discrepancy between thedegree of albumin excretion rate and the clearance of36-A Ficoll molecules may reflect impaired albuminreabsorption by the proximal tubule. Another possibleexplanation is that the macromolecular shape of Ficollmolecules during gbomerular ifitration may differ

Effective Molecular Radius (A)

Figure 4. FractIonal clearance of Ficoll of graded size dlstri-bution determined on the baseline (0 to 10 mm perfusiontime; Basal) and the last experimental clearance (50 to 60mm perfusion time; All) samples during All Infusion In thepresence of the specIfic All receptor antagonist SR 47436 (8ng/mmn) In Isolated perfused rat kidney.

enough from that of albumin to make it more perme-able, in spite of similar permeability characteristicsunder tn vttro conditions.

The data from this study in isolated perfused kid-ney, and previous findings tn vtvo in the rat, have beeninferred to signify a role of the renin-angiotensinsystem in the pathogenesis of the barrier dysfunction

that underlies gbomerular proteinuria in humans.However, Loon and coworkers (42) have recentlyshown that in healthy humans, in striking contrast tothe rat, pressor All infusion had some influence onbarrier size-selectivity, as documented by the in-creased fractional clearance of neutral dextrans overthe entire radius interval measured (34 to 54 A), but

Laplnski et al

Journal of the American Society of Nephrology 659

actually lowered the level of albuminurla. The antipro-

teinuric effect exerted by All has been attributed to the

concomitant reduction of the ifitered protein loadbecause of a prompt fall in GFR. As possible explana-

tions for the disparate effect of All infusion on theifitered protein load in rats and humans, infusion ofpharmacobogical rather than physiological doses of All

in the rat (35,36,43), as well as differences in thedynamics of gbomerular ifitration between species

(35,36,43) and the peculiar conditions of isolatedperfused kidney, have to be taken into account. Thus,some caution should be exercised before extrapolating

our findings in this study with a preparation withaltered ifitration properties, such as isolated kidney,to the intact human kidney.

In conclusion, our study indicates that in isolated

perfused kidney: (1 ) All infusion induces a progressiveand dose-dependent increase in urinary protein ex-

cretion rate and loss of gbomerular size-selective func-tion, with or without an increase in FF, depending on

the rate of infusion of All that was used; (2) All-

induced permselectivity defects are completely pre-vented by a selective Al! Type 1 receptor antagonist;(3) All-induced proteinuria is not completely explainedby the observed defect in glomerular size-selectivity.

and a concurrent specific alteration of charge-selec-tivity is very likely to contribute.

ACKNOWLEDGMENTSWe thank Sanofi Recherche, Montpellier, France for kindly providing

SR 47436 throug�i the courtesy of Dr. Dino Nisato.

REFERENCES1 . Anderson S, Meyer TW, Rennke HG, Brenner BM: Con-

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7. Marre M, Le Blanc H, Suarez L: Converting enzymeinhibition and kidney function in normotensive diabeticpatients with persistent microalbuminurla. Br Med J1987;294: 1448-1452.

8. Parving HI!, Hommel E, Smidt UM: Protection of kidneyfunction and decrease in albuminuria by captopril ininsulin dependent diabetics with nephropathy. Br Med J1988;297: 1086-1091.

9. Anderson S, Rennke G, Garcia DL, Brenner BM: Shortand long term effects of antihypertensive therapy in the

diabetic rat. Kidney mt l989;36:526-536.10. Perlco N, Amuchastegul CS, Malanchini B, Bertani T,

Remuzzi G: Angiotensin-converting enzyme inhibitionand calcium channel blockade both normalize earlyhyperifitration in experimental diabetes, but only theformer prevents late renal structural damage. Exp Neph-rob 1994;2:220-228.

1 1 . Remuzzi A, Imberti 0, Puntorieri 5, et at.: Dissociationbetween antiproteinuric and antihypertensive effect ofanglotensin converting enzyme inhibitors in rats. Am JPhysiol 1994;267:Fl034-F1044.

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15. Lebovitz HE, Wiegmann TB, Cnaan A, et aL: Renalprotective effects of enalapril in hypertensive NIDDM:Robe ofbasellne albuminuria. Kidney Int 1994;45(Suppl45):S150-5155.

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19. Myers BD, Guasch A Mechanisms of massive protein-uria. J Nephrol l994;7:254-260.

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collagen molecules in basement membranes. Eur J Bio-chem 1981;120:203-211.

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40. Shake JG, Brandt RC, Daniels BS: Angiotensin II in-duces actin polymerization within the glomerular ifitra-tion barrier: possible role in the local regulation ofultrafiltration [Abstracti. J Am Soc Nephrol 1992;3:568A.

4 1 . Oliver JD Ill, Si.mons JL, Troy JL, ProvoostAP, BrennerBM, Deen WM: Proteinuria and impaired glomerularpermselectivity in uninephrectomized fawn-hooded rats.Am J Physiol 1994;267:F917-F925.

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My Dear Starling,I have now completed the task which you allotted me in your capacity as editor of this series and which I have

found more burdensome than I expected. The growth in the literature on the kidney has been extraordinary since thetime when you and I began to work on it, and this increase in bulk has not gone along with an improvement ofquality, but rather the reverse. No other organ of the body has suffered so muchfrom poor work as the kidney, andin no other region ofphysiology does so much base coin pass as legal tender I do notfiatter myse�fthat thistreatise attainsfinality in regard to the secretion of the urine: that is impossible at the present time. If it serves as

an advanced postfrom which others may issue against the remaining ramparts of vitalism, its purpose will beattained.

Arthur R. Cushny: from a letter to Ernest H. Starting which appears as a perface to “The

Secretion of the Urine.” Longmans, Green and Co. Ltd., London. 1917.