Nephrol Dial Transplant (1996) 11: 2163-2169

Original Article

NephrologyDialysis

Transplantation

Inhibition of aminopeptidase A activity causes an acute albuminuria inmice: an angiotensin ll-mediated effect?

S. Mentzel1, K. J. M. Assmann1, H. B. P. M. Dijkman1, A. S. de Jong1, J. P. H. F. van Son1,J. F. M. Wetzels2 and R. A. P. Koene2

Departments of 'Pathology and 2Internal Medicine, Division of Nephrology, University Hospital Nijmegen,6500 HB Nijmegen, The Netherlands

Abstract The hydrolase aminopeptidase A is animportant regulator of the renin-angiotensin system,since it inactivates its most vasoactive componentangiotensin II (Ang II). A single i.v. injection of amonoclonal antibody against mouse aminopeptidase A(ASD-4) induces a membranous-like glomerulo-nephritis in mice, characterized by an acute albumin-uria, that is not dependent on complement, thecoagulation system, or inflammatory cells. We hypo-thesized that this albuminuria is the consequence of areduction in aminopeptidase A enzyme activity, thatmight subsequently lead to an increase in Ang II levels.Aminopeptidase A enzyme activity was analysed invitro by a fluorimetric enzyme assay and in vivo byenzyme histochemistry. The role of Ang II in theinduction of albuminuria in this model was studied bymeasuring the renal aminopeptidase A mRNA expres-sion in our model by a competitive PCR assay as anindirect measure of Ang II levels. In addition, the roleof Ang II in this model was studied by preventing theformation of Ang II with the angiotensin-convertingenzyme inhibitor enalapril or by blocking of the Ang IIreceptor with the ATI receptor antagonist losartan.Only antibodies that were able to inhibit the amino-peptidase A enzyme activity in vitro and in vivo inducedan acute albuminuria in mice. Renal aminopeptidase AmRNA expression was increased by injection of theanti-aminopeptidase A antibody. Both enalapril andlosartan treatment reduced the acute albuminuria,measured 1 day after injection of a monoclonal anti-body against aminopeptidase A, by 91% and 83%,respectively. It is concluded that the induction of acutealbuminuria is correlated to the enzyme-inhibitingcapacity of the anti-aminopeptidase A antibodies. Thisimpaired enzymatic activity most likely leads to anincrease in the levels of Ang II, the best knownsubstrate of aminopeptidase A. The results of ouradditional experiments are in keeping with our hypo-thesis that Ang II mediates this acute albuminuria.

Correspondence and offprint requests to: S. Mentzel, Department ofPathology, University Hospital Nijmegen, P.O. Box 9101, 6500 HBNijmegen, The Netherlands.

Whether this occurs by an increase of blood pressureor by a growth factor-like effect remains to be definedby further studies in this model.

Key words: membranous glomerulonephritis; amino-peptidase A; albuminuria; renin-angiotensin system;angiotensin II; mouse

Introduction

Aminopeptidase A (APA; EC 3.4.11.7) is a homodi-meric, type II integral cell membrane-associated glyco-protein with a molecular weight of approximately140 kDa of each monomer. APA has a widespreadorgan distribution, but in the mouse kidney it ispredominantly expressed on podocytes and brush bor-ders of proximal tubular epithelial cells [1,2]. In gen-eral, APA hydrolyses amino-terminal glutamyl andaspartyl residues from peptides [3]. The best knownbiological function of this enzyme is related to its effecton the renin-angiotensin system. By cleaving theN-terminal aspartate from angiotensin II (Ang II),APA converts the most active component of the renin-angiotensin system to the less active component Ang III[4]. Apart from this prominent role in the renin-angiotensin system, its biological function is largelyunknown [5].

Circulating antibodies can bind to podocyticenzymes such as APA. The interaction of antibodieswith their antigenic targets on the cell membranes ofpodocytes is one of the mechanisms that underlies theformation of subepithelial immune complexes and is ahistological hallmark of membranous glomerulo-nephritis [6]. Recently, we have reported that a singlei.v. injection of a monoclonal antibody (mAb) againstAPA into mice induced a membranous-like glomerulo-nephritis, that was accompanied by an acute albumin-uria. The induction of this acute albuminuria was notdependent on systemic mediators of inflammationknown from other experimental models of glomerulo-nephritis [1]. In view of the fact that APA plays such

© 1996 European Renal Association-European Dialysis and Transplant Association

2164

a prominent role in the degradation of Ang II, wehypothesized that the binding of the mAb to APAmight inhibit the APA enzyme activity, and sub-sequently might lead to a reduced inactivation andconsequent accumulation of Ang II. We speculatedthat an enhanced or prolonged effect of Ang II onsystemic or glomerular haemodynamics, renal cellgrowth, or renal cytokine production might be respons-ible for the induction of acute albuminuria in thismouse model. This hypothesis was tested by determin-ing the relationship between the occurrence of albumin-uria and the ability of the mAb to inhibit APA enzymeactivity in vitro and in vivo. Furthermore, we havemeasured renal APA mRNA expression as a markerof Ang II activity, and have examined the effects ofthe angiotensin-converting enzyme (ACE) inhibitorenalapril and the Ang II type 1 ATI receptor antagonistlosartan on the induction of acute albuminuria.

Subjects and methods

Animals

BALB/c and BALB/c, nu/nu mice, weighing 20-25 g andaged 3-6 months, were originally obtained from the JacksonLaboratory (Bar Harbor, ME). All mice were bred in thebreeding facility of the central animal laboratory of ouruniversity by continuous brother-sister matings.

Monoclonal antibodies

A rat mAb against mouse APA (ASD-4) used for most ofthe studies was raised as described [1], and propagated asascites in BALB/c, nu/nu mice. Isolation of the rat mAbfrom ascites was done by ammonium sulphate precipitation.Seven other rat mAbs against APA used in this study (ASD-2,ASD-3, ASD-37, ASD-38, ASD-39, ASD-41 and ASD-44)were generated and purified in the same way. The concentra-tion of the rat mAb was determined by radial immunodiffu-sion [7], The effect of the mAb on the APA enzyme activitywas tested in vitro by preincubation of the mAb with a brushborder-enriched renal suspension [1].

Enzyme histochemistry

The enzyme activity of APA in the kidneys was visualizedby enzyme histochemistry according to Lojda and Gossrau,as described before [2,8].

APA mRNA in situ hybridization

Parts of the kidneys frozen in liquid nitrogen were used fordetection of APA mRNA by RNA in situ hybridization usingboth a sense and antisense 344 bp digoxigenin-labelled cRNAprobe, as described before [9].

Competitive PCR assay for APA mRNA

To compare the expression of APA mRNA under the differ-ent experimental procedures, we measured the APA mRNAconcentrations extracted from a part of one kidney, with acompetitive polymerase chain reaction (PCR) assay.

S. Mentzel et al.

Construction of competitor molecule

Oligonucleotides were synthesized using the 380B DNAsynthesizer (Applied Biosystems, Foster City, USA), andprimers were selected on the basis of the mouse APAcDNA sequence [10] using the PRIMER software program(Version 0.5, MIT, Cambridge, MA). For the generation ofthe competitor molecule as an internal standard a PCR wascarried out on the II 1-6 mouse APA cDNA clone (kindlyprovided by M. D. Cooper, Birmingham, AL [10] using thefollowing primers: ON-1: 5'-TGG AGA GAG CCT CGGCTA TCC AAT CCC ACG TTC C-3', complementary tonucleotides 1959 to 1993 and ON-14: 5'-TTC ACA TCCAGT GTT CGT CAG GGG GAG CAA GGG AGC TTCTAT TCT-3'. The 5' primer ON-14 was synthesized to allowpriming with its 5' part to nucleotides 1430 to 1450 of themouse cDNA sequence and priming with its 3' part tonucleotides 1502 to 1521, skipping 52 base pairs. In addition,a five base pair spacer was added to this ON-14 primer toallow proper folding of the complementary strand. The PCRmixture contained 65 mM KC1, 20 mM Tris-HCl pH 8.8,4.8 mM MgCl2, 2 mM DTT, 0.1% NP-40, 0.25 mM dNTPeach, 500 nM ON-1, 500 nM ON-14, Ing II 1-6 cDNA,0.25 U Supertaq in a 100 ul volume. Reactions were overlaidwith two drops of mineral oil. Amplification in a DNAthermal cycler (Perkin Elmer Cetus, Norwalk, CT) startedwith an initial denaturation step at 94°C for 5 min followedby 40 cycles at 94°C, 42°C, and 72°C each for 1 min. The509 base pair PCR product was purified using the WIZARD®PCR preps DNA purification system, and analysed on anethidium bromide-stained 2% agarose gel. The DNA concen-tration and purity were measured using the Genequant®DNA/RNA calculator (Pharmacia, Uppsala, Sweden).

Competitive assay

Total cellular RNA of half a kidney of mice (n = 5) from thedifferent experimental groups, snap frozen in liquid nitrogenand homogenized with a micropestle, was isolated using theRNAzol B method [11]. The isolated RNA was controlledby agarose gel electrophoresis for integrity, and measuredand checked for purity by optical density in a Genequant®DNA/RNA calculator (Pharmacia, Uppsala, Sweden). Forthe competitive RT-PCR assay 1 ug of isolated total cellularRNA was reverse transcribed for 1 h at 37°C using the ON-1primer in the following RT mixture (20 ul): 75 mM KC1,50 mM Tris-HCl pH 8.3, 6mM MgCl2, 10 mM DTT,0.2 mM dNTP each, 1.25 uM ON-1, and 4.5 U AMV reversetranscriptase. After completion of the RT reaction 20 ul ofa serial dilution in distilled water of the II 1-6 competitormolecule ranging from 1.3 x 10~18 to 9.6 x 10~17 mol wereadded to these samples. Then 60 ul of PCR mixture wereadded giving the final concentrations of 65 mM KC1, 20 mMTris-HCl pH 8.8, 4.8 mM MgCl2, 2 mM DTT, 0.1% NP-40,0.3 U Supertaq, 500 nM ON-1, and 500 nM ON-2: 5'-TTCACA TCC AGT GTT CGT CA-3'. The ON-2 primer isidentical to the 5' part of ON-14 and complementary tonucleotides 1430 to 1450 of the mouse APA cDNA sequence,so that both the competitor molecule and the DNA/RNAhybrid reverse transcribed from APA mRNA can be ampli-fied. The final samples of 100 ul were overlaid with twodrops of mineral oil, and after an initial denaturation stepat 94:C for 5 min, the mixture was amplified for 35 cycles at94°C, 55aC, and 72°C each for 1 min. Twenty ul of the PCRproduct were analysed on an ethidium bromide-stained 2%agarose gel. To control whether the competitive PCR yielded

Inhibition of renal aminopeptidase activity causes an acute albuminuria in mice 2165

quantitatively reproducible results, a known concentrationof the II 1-6 cDNA insert (2.7 xlO"18 mol/reaction) wasadded instead of a constant concentration of cDNA obtainedby RT from total renal RNA (Table 2).

After electrophoresis the agarose gel was uniformly lit byan ultraviolet light source. The fluorescence signal was digit-ized using a CCD RGB camera with a macro-objectiveattached to a VIDASplus image analysts system. Using regres-sion analysis the value at log ratio 0 could be calculated,resulting in the value corresponding to the concentration ofthe mRNA.

Effect of enalapril and losartan treatment

The effect of the ACE inhibitor enalapril and the ATIreceptor blocker losartan on the acute albuminuria inducedby injection of a mAb against APA (ASD-4) was examinedin six groups of BALB/c mice. The effective dosing scheduleof enalapril was determined in a pilot study in which weassessed the ability of giving enalapril by gavage or in thedrinking water to attenuate serum ACE activity. For losartan,we adopted a comparable dosing schedule. Three groupsreceived 8 mg ASD-4 alone (B), or in combination withenalapril (D) or losartan (F). Three control groups receivedthe same volume of saline instead of ASD-4 alone (A), orin combination with enalapril (C) or losartan (E). Treatmentwith enalapril (C and D) or losartan (E and F) started 24 hbefore the administration of ASD-4 or saline by adding100 mg/1 enalapril or losartan to the drinking water and wascontinued for the following 2 days. Additionally, every 12 hall groups were given 0.5 mg enalapril (C, D) or losartan(E,F), or the same volume of drinking water (A, B) bygavage. Six h after the i.v. injection of ASD-4 or saline, micewere placed in a metabolic cage as described [1,12] and urinewas collected during an 18 h period. Twenty-four h after thei.v. injections blood was collected and mice were killed bycervical dislocation, and their kidneys removed and processedfor light microscopy, immunofluorescence, RNA in situhybridization, and RNA isolation. Albuminuria, defined asthe amount of albumin excreted during 18 h (mean + SEM),as a sign of glomerular protein leakage, was determined by

Table 1. Characteristics of eight mAbs against mouse aminopeptid-ase A

Code mAb

ASD-3ASD-4ASD-37ASD-39

ASD-2ASD-38ASD-41ASD-44

Albuminuria atday I1

9068 + 26107494 + 28775951+9905948+1343

96 + 34120 + 46133 + 14120 + 62

FEA (%]

8101822

9611693

105

Enzyme histochemistry

I2 Glomeruli Proximaltubules

+++±+ + + + + ++ + + + + ++ + + + + ++ + + + + +

1 For the mAb ASD-37 and ASD-39 20 mg was injected instead ofthe 10 mg for the other mAb. Data are expressed as ug of albuminexcreted per 18 h. 2Residual APA activity of a brush border-enrichedrenal suspension after preincubation with 100 ug of each mAb in thefluorimetric enzyme assay (FEA).

radial immunodiffusion using a goat anti-mouse albuminantiserum as described [7,12].

Light microscopy

Kidney fragments were fixed in Bouin's solution, dehydrated,embedded in paraplast, and 2 (im sections were stained withperiodic acid Schiff, and silver methenamine, as describedearlier [1].

Statistical analysis

For statistical analysis, one-way analysis of variance(ANOVA) was used and group comparisons were done withthe Tukey-Kramer test. P values < 0.05 were regarded assignificant. All values are expressed as means + SEM.

Results

Induction of acute albuminuria

Eight rat mAbs against the mouse hydrolase APA weredeveloped in our laboratory [1]. A single i.v. injectionof these mAbs in normal mice resulted in a clear-cut,acute albuminuria using the mAbs ASD-3, ASD-4,ASD-37 and ASD-39, although ASD-37 and ASD-39had to be given in higher doses (Table 1). The fourother mAbs (ASD-2, ASD-38, ASD-41 and ASD-44)never induced albuminuria, even at higher doses(Table 1).

Effects on APA enzyme activity

The ability of the anti-APA mAbs to inhibit the APAenzyme activity was tested on a solubilized brushborder suspension. The strongly albuminuric mAbsASD-3 and ASD-4 inhibited the enzyme activity byapproximately 90%, the somewhat less effective mAbsASD-37 and ASD-39 inhibited the enzyme activity byapproximately 80%, while the non-albuminuric mAbsASD-2, ASD-38, ASD-41 and ASD-44 had no effecton the APA enzyme activity in vitro (Table 1). Inaddition, enzyme histochemistry on frozen sectionstaken 1 day after injection of the eight mAbs showedthat the renal APA enzyme activity was almost com-pletely inhibited when the mAbs ASD-3, ASD-4,ASD-37 or ASD-39 were injected, while the four non-albuminuric anti-APA mAbs had no effect on theenzyme activity (Table 1). ASD-4 was used for furtherexperiments. The effect of this mAb on the renal APAenzyme activity is illustrated in Fig. 1. One day afterinjection of ASD-4 the renal APA enzyme activity wasalmost completely inhibited, except for a minute glom-erular enzyme activity (Fig. 1A), while no effect onthe enzyme activity was seen in saline-injected controlmice (Fig. IB).

APA mRNA alterations

To further delineate the role of Ang II in the inductionof albuminuria in our model we tried to quantitatetissue Ang II levels. Unfortunately, we were not ableto measure tissue Ang II levels directly. However, it

2166 S. Mentzel el al.

Fig. 1. Enzyme histochemistry on frozen kidney sections of a BALB/c mouse 1 day after i.v. injection of 8 mg ASD-4 (A) or saline (B,control). (A) Injection of ASD-4 almost totally abolished the enzyme activity in the entire kidney of the mouse. Only very faint activitycan be seen in the glomerulus (x480). (B) In the control mouse APA activity is present in the glomerulus and in the brush borders of theproximal tubular cells (x480).

Fig. 2. Expression of APA mRNA in a normal BALB/c mouse kidney by non-radioactive RNA in situ hybridization (RISH). (A) RISHusing the antisense riboprobe for APA showed strong cytoplasmie staining of cells located at the periphery of the gloraerulus (arrows).Only a faint or negligible staining can be seen in the proximal tubular epithelial cells (x 1000). (B) RISH with the sense riboprobe for APAwas completely negative (x 1000).

Table 2. Pilot experiments to test the accuracy and reproducibilityof the competitive PCR

Sample Number of Correlation cDNA concentrationnumber regression points coefficient (amol/sample)

123

161616

0.980.960.98

2.72.73.0

To test the accuracy and reproducibility of the competitive PCRassay, 2.7 amol of the II 1-6 cDNA clone, coding for mouse APA,were co-amplified with varying amounts of competitor molecules asdescribed in the Methods. A variation of 4.3% between the measuredAPA cDNA concentration and the known input cDNA concentra-tion was found.

22< ts

Inhibition of renal aminopeptidase activity causes an acute albuminuria in mice

has been demonstrated that Ang II induces renal APAexpression. Kidney APA mRNA levels might therefore ^provide an indirect measure of renal Ang II. We have g ^determined kidney APA mRNA by in situ hybridiza- '« |£tion and by a more sensitive technique, the competitive « 2PCR assay. g- ~RNA in situ hybridization. Non-radioactive RNA insitu hybridization with an antisense riboprobe for APAshowed strong staining of glomerular cells, and a faintor negligible staining of proximal tubular epithelialcells and smooth muscle cells of arteries in normalmice (Fig. 2A). RNA in situ hybridization at theelectron microscopic level demonstrated that in theglomerulus APA mRNA was confined to the podo-cytes, and not to the endothelial or mesangial cells(data not shown). Hybridizations with the sense ribop-robe were negative (Fig. 2B). Using RNA in situhybridization we did not observe differences in expres-sion between the different experimental groups.Competitive PCR assay. To examine slight variationsin APA mRNA expression in the different experimentalgroups 1 day after induction of the anti-APA glom-erulonephritis, we used a competitive PCR assay thatwas able to measure attomol levels of APA mRNAfrom a part of a single kidney of normal mice. Tocontrol whether the competitive PCR assay followingthe RT step gave reproducible, quantitative results, theassay was carried out with the II 1-6 APA cDNAinsert of known concentration instead of a constantconcentration of reverse transcribed RNA. In threeseparate experiments we found only a minor divergence(4.3%) from the known concentration of added cDNA,making this assay a very reproducible, sensitive andaccurate procedure with a correlation coefficient of>95%(Table2).

Mice injected with ASD-4 (group B) showed a1.7-fold increase in APA mRNA compared to controlmice (group A) receiving saline (36.5 + 1.8 vs20.6 + 2.1 amol APA mRNA/ug total RNA; P<0.05)(Fig. 3). Enalapril treatment (group D) considerablyreduced the APA mRNA expression induced by ASD-4(25.4+ 3.0 amol APA mRNA/ug total RNA).Enalapril treatment alone (group C) had no influ-ence on APA mRNA levels (22.2 ±2.6 amol APAmRNA/ug total RNA). Losartan treatment alone

2167

50-

<o 2 5 -

Saline ASD-4 Saline ASD-4 Saline ASD-4

enalaprS enalapril losartan losartan

Fig. 3. Measurements of APA mRNA levels during induction ofacute albuminuria and interventions with enalapril and losartanusing a competitive PCR assay. The groups analysed by this compet-itive PCR assay are the same as in Table 3. Values are expressed asamol/ug of total cellular RNA and are given as means + SEM.*P<0.05, **P<0.00\ compared to group with saline and water.

(group E), or in combination with ASD-4 (group F),had a 2.3-fold enhancing effect on the APA mRNAlevels compared to control mice (group A) (49.2 + 5.7and 48.1+9.3 amol APA mRNA/ug total RNArespectively; P<0.001).

Reduction of tlie anti-APA induced acute albuminuria byenalapril and losartan

To further support the hypothesis that Ang II isinvolved in the induction of acute albuminuria byadministration of anti-APA mAb, both the ACE inhib-itor enalapril and the ATI receptor antagonist losartanwere given in two separate experiments. Enalapril andlosartan treatment was started 1 day before administra-tion of ASD-4 to ascertain that the ACE activity andATI receptor were completely blocked at the timeASD-4 was given. One day after administration ofASD-4, the albuminuria induced by ASD-4 wasreduced by 91% and 83% in the enalapril and losartan-treated mice, respectively (Table 3). The albuminuria

Table 3. Effect of enalapril and losartan treatment on the acutealbuminuria at day 1 after injection of ASD-4'

Group n Enalapril2 Losartan2 ASD-4 Albuminuria3

(ug/18h)

ABCDEF

161611111010

93± 182717±872

98±5259±1804

77±9448 ±124"

1 Eight mg of ASD-4 or the same volume of saline (controls) wasgiven i.v. 2 Enalapril or losartan was given by drinking water(100 mg/l) and every 12 h by gavage(0.5 mg). Control mice receivedthe same volume of drinking water by gavage. 3Mean + SEM.4/J<0.005 compared to group A.

2168

of mice of the control groups, given saline with orwithout enalapril or losartan, did not exceed the upperlimit of the physiological albuminuria (< 100 ug/18 h).(Immuno-) histological findings. By light microscopy,mice treated with ASD-4 showed no glomerular abnor-malities. The cells of the convoluted and straight partsof the proximal tubules were finely vacuolated andoccasionally protein casts were seen. These were absentin ASD-4-injected mice treated with enalapril andlosartan (data not shown). Immunofluorescence at 1day after injection showed binding of ASD-4 in allexperimental groups to the glomerular capillary wallin a homogeneous or fine granular pattern as reportedearlier [1]. Treatment with enalapril or losartan hadno effect on this binding of ASD-4.

Discussion

In several forms of experimental membranous glom-erulonephritis, induced by the binding of antibodies tointrinsic antigens on podocytes, it has been shownthat podocytes are not just innocent bystander cells.The interaction of antibodies with their targets onthe podocytes induces the release of several pro-inflammatory and vasoactive substances that caninfluence glomerular permeability and play a role inthe progression of the glomerular lesions [6,13]. Oneof these podocytic antigens in experimental forms ofmembranous glomerulonephritis is the hydrolase APA.Recently, we have reported that injection of a mAbagainst APA in mice induced a membranous-like glom-erulonephritis that was characterized by a dose-dependent, acute albuminuria. However, in contrast tomost other experimental models, this acute albumin-uria was not mediated by an inflammatory response[1]. Theoretically, several other mechanisms might beinvolved in the induction of the acute albuminuria inour model. First, binding of the mAb to the podocytemight trigger the release of one or more mediators,that are capable of altering glomerular permeability.Second, albuminuria might be related to an inhibitedactivity of APA, and subsequently reduced degradationof one of its substrates. In the present study we havefocused on this latter possibility.

Our data clearly demonstrate that only the mAbsthat inhibit APA enzyme activity can induce albumin-uria. This finding suggests that APA is not merely atarget on the podocyte but that the APA enzymeactivity plays a crucial role in the induction of acutealbuminuria in this model. The involvement of an APAsubstrate molecule seems likely. However, only a fewpossible substrates have been identified. The bestknown substrate of APA is Ang II, the most activecomponent of the renin-angiotensin system. Ang II ofendogenous or exogenous origin has long been knownto increase urinary protein excretion in various experi-mental settings [14], which can be explained by amarked elevation of the glomerular capillary pres-sure and/or systemic blood pressure. Alternatively,increased local or systemic Ang II levels might activate

S. Mentzel el al.

renal cells to release growth factors and cytokines, thatindirectly can modulate the glomerular permselectivity.

To confirm that inhibition of APA enzyme activitycauses proteinuria by increasing Ang II levels, we triedto quantitate tissue Ang II levels. Unfortunately, wewere unable to directly measure mouse tissue Ang IIlevels. Therefore, we concentrated on methods whichindirectly reflect Ang II levels, such as measurementsof the Ang II receptor or expression of renin or APA[15-19]. It has been shown that increasing Ang IIlevels by either exogenous (infusion of Ang II) orendogenous routes (rat remnant kidney model)increases the renal APA expression [17-19]. Enzymehistochemistry cannot be used in our model sinceASD-4 completely blocks the APA enzyme activity invivo. By in situ hybridization we observed a clear APAmRNA expression in the podocytes, but only faintexpression in the proximal tubular epithelial cells. Sucha difference in tubular and glomerular APA mRNAexpression has also been observed in the rat [20]. Thisfinding is not in correspondence with the immunoreac-tive pattern shown in earlier studies [1,2,21]. Thisdifference is certainly not due to alternative splicingphenomena or different isoforms, since only a singleAPA mRNA species can be identified in mouse tissues[2,21]. The difference between immunoreactivity andAPA mRNA expression might be explained by a lowturnover of APA in the proximal tubular epithelialcells, when compared to the podocytes. In situ hybrid-ization proved insensitive to detect changes in APAmRNA expression. Therefore, we measured the APAmRNA expression by a sensitive, competitive PCRassay that produced accurate and reproducible results.The administration of ASD-4 resulted in a 1.7-foldincrease in APA mRNA expression, which is in linewith our hypothesis that blockade of APA enzymeactivity increases the Ang II concentration. Enalapril,which reduces the generation of Ang II, lowered theAPA mRNA expression. The ATI receptor blockerlosartan when given alone increased APA mRNAexpression. This result is in agreement with the increasein Ang II levels which is invariably seen during ATIreceptor blockade [22]. This also indicates that theAng II-induced increase of APA expression is notmediated through the ATI receptor. Our observationsfit with the findings that the ATI receptor is locatedon mesangial cells and spatially separated from APA,which is localized only on the podocytes. This is alsosupported by the finding that the expression of APAin podocytes is mediated by increases in intracellularcAMP, a second messenger that is not coupled to theATI receptor [23,24].

We further examined the role of Ang II in our modelby preventing its generation by ACE inhibition withenalapril. This drug has successfully been used as ananti-hypertensive and anti-proteinuric agent in humansand experimental animal models [25]. However, theeffects of enalapril may not solely have been due toinhibition of the generation of Ang II; enalapril alsoretards the breakdown of the vasodilator bradykinin,which may lead to a decrease of blood pressure and

Inhibition of renal aminopeptidase activity causes an acute albuminuria in mice 2169

proteinuria [25,26]. Therefore, we also prevented thebinding of Ang II to its ATI receptor with losartan, anon-peptide ATI receptor antagonist [27]. ATI recep-tors are known to mediate all known haemodynamicand proliferative effects of Ang II [28]. Both enalapriland losartan considerably reduced the acute albumin-uria in our anti-APA model (by 91% and 83%, respect-ively), strongly suggesting that Ang II plays animportant role in the development of acute albuminuriain our model. We cannot exclude, however, that thedecreased proteinuria is a consequence of a non-specificreduction in blood pressure.

The anti-APA glomerulonephritis is a model ofpassive membranous-like glomerulonephritis in mice,in which the antigenic target is functionally and mor-phologically well denned. We have shown that theoccurrence of albuminuria depends on the ability ofan mAb to block APA enzyme activity, that the expres-sion of renal mRNA for APA increases, and that bothenalapril and losartan reduce albuminuria. All thesedata strongly suggest that Ang II plays an importantrole in the pathogenesis of the acute albuminuriaoccurring in this model. Our model and thedeveloped mAb will enable further studies on the roleof APA and Ang II in renal injury.

Acknowledgements. This study was supported by a grant from theDutch Kidney Foundation (C91.1169). We thank J. Koedam for hisassistance with the animal experiments, and J. van de Laak for hisassistance with the VIDASplus image analyser. We also thank DrCooper from the University of Alabama (Birmingham, USA) forproviding us with the mouse APA cDNA clone. Enalapril was a giftof Merck-Sharp-Dome (Rahway, USA). Losartan was kindly pro-vided by DuPont (Wilmington, USA).

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Received for publication: 3.4.96Accepted in revised form: 10.7.96