University of Groningen

Towards an integrated approach on RAAS-blockadeLely, Anna Titia

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ANGIOTENSIN CONVERTING

ENZYME GENOTYPE IN WISTAR RATS

Titia LelyHilde Tent

Harry van GoorGerjan Navis

Ron Korstanje

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AbstractIn humans, the insertion/deletion polymorphism in the angiotensin converting enzyme (ACE) gene

accounts for half of the variance in plasma ACE activity. The deletion allele is associated with

cardiovascular and renal disease, but analysis of the mechanisms is difficult due to complex genetic

and environmental interactions. Identification of a microsatellite marker in intron 13 of the rat ACE

gene, on chromosome 10 has allowed differentiation of ACE alleles between the Brown Norway (B)

and Lewis (L) strains and has shown phenotypic differences between rats carrying the B and L alleles.

However, no data are available on variation of the gene within an outbred rat strain. Therefore, we

investigated the role of the ACE gene in the outbred Wistar rat.

In the outbred Wistar rat the B/L variation was identified. Subsequently, two Wistar lines

homozygote BB and LL were set up. Urinary protein excretion was measured in 12 week old rats

(n=40). Afterwards, rats were sacrified and ACE activity (Hip-His-Lue cleavage) and mRNA expression

were measured in heart, kidney, and aorta homogenates. In serum, ACE activity was measured.

Immunohistochemistry for ACE was performed in the heart, kidney and aortic tissue. In 6 week old rats

(n=14) aortic tissue was collected. The vasoconstrictor responses to ang I and ang II were studied in

aortic rings.

Serum, heart, and kidney ACE activity and kidney mRNA expression were two-fold higher in Wistar

BB (all p<0.05). Remarkably, in aorta ACE activity was two-fold higher in Wistar LL (BB: 147 (122-199)

vs LL: 310 (291-360) nmol/His-Leu/mL/min, p<0.05). However, functional conversion of ang I was

higher in Wistar BB (area between ang I and ang II curves BB: 2 (-31-37) vs LL: 69(33-95), p<0.05),

indicating functional difference in ang I to ang II conversion between the two alleles. Finally, kidney

staining showed a clear difference between tubular ACE expression with more staining in Wistar BB.

Urinary protein excretion was similar (BB: 20(17-26) vs LL: 19(17-26)), ns).

We conclude that the Wistar B/L genotype lines, bred and characterized in this study, offer

interesting opportunities to study the effects of genetic differences in ACE levels on renal and

cardiovascular pathophysiology.

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IntroductionIn human, the insertion/deletion (I/D) polymorphism of the ACE gene, accounts for half of the

variance of circulating and tissue ACE levels, with the highest ACE levels in DD homozygotes, the

lowest in II homozygotes, and intermediate values in heterozygotes [1]. The D allele of the ACE

genotype has been associated with cardiovascular and renal disease [2;3]. However, the results are

not uniform and analysis of both its pathophysiological significance and the underlying mechanisms is

complicated by multiple interactions with other genetic and environmental factors [4;5].

Identification of a microsatellite marker in the rat ACE gene, specifically in intron 13, has allowed

differentiation of ACE alleles among different rat strains [6;7], and their association with different levels

of plasma and tissue ACE. The ACE locus determines 74% of the plasma ACE activity in Brown

Norway and Lewis rats after cross-breeding, where the B allele is related to high plasma ACE activity

and the L allele to low plasma ACE activity [6;8].

Challah et al. demonstrated that ACE activity in cultured vascular cells and the neointima formation

in the carotid artery after balloon injury were influenced by ACE levels [8]. Moreover, Ocaranza et al.

showed there is an enhanced hypertensive response and more fibrosis in response to myocardial

infarction in the Brown Norway rat with higher ACE levels [9;10]. When studying the effect of the B

and L alleles and their difference in ACE expression on the phenotype, it is important to realize that the

Brown Norway (B) and Lewis (L) rat strains have completely different genetic backgrounds. Therefore,

variations in other genes might have influenced the difference in susceptibility to damage.

In outbred Wistar rats, individual differences in renal ACE activity are independent predictors of the

susceptibility to renal damage [11]. To provide a better model to study the human ACE I/D genotype,

we investigated the ACE genotype in the outbred Wistar rat and its relation to tissue and serum ACE

activity (Hip-His-Leu cleavage), and bred two strains with respectively high and low genetically

determined ACE activity. Moreover, we investigated whether there the difference in ACE alleles lead to

a functional difference conversion of ang I to ang II in the vascular wall. To this purpose, we

investigated the contractile response to ang I in reference to the response to ang II in isolated aortic

rings, as a functional assessment of vascular ACE activity.

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Materials and Methods

Animals Wistar (HsdCpb:WU) rats were obtained from Harlan (Harlan Inc, Horst, The Netherlands) and

genotyped for the ACE allele. Animals were then selected to breed 27 homozygous males for both the

B allele and the L allele. All animals were housed in a climate controlled space with a 14h light / 10h

dark cycle. Food and water were available ad libitum. After six weeks, 14 rats (seven for each allele)

were anaesthetized using Isoflurane/O2/N20 mixture. Thereafter, aortic tissue was collected and

immediately placed in Krebs bicarbonate solution (compound from Merck, Darmstadt, Germany). At 12

weeks, the other 40 rats were placed in metabolic cages; 24h urine samples were collected.

Afterwards rats were sacrificed and renal, heart and aortic tissue and blood samples were collected.

The abdomen was opened through midline incision. A two-ml blood sample was obtained via aortic

puncture for determination of serum ACE activity. Kidneys were saline perfused and harvested. Renal

cortical tissue from the upper pole was processed for ACE determination. Mid-coronal renal tissue

slices were processed for immunohistochemical examination. All animal experimentation was

approved by the University’s Animal Care and Use Committee. The study was conducted in accord

with the NIH Guide for the Care and Use of Laboratory Animals.

Genotyping Genomic DNA was isolated from tail tips as previously described [12]. To determine the ACE

genotypes, primers were used as described by Hilbert et al. [6]. They amplify the microsatellite located

at the 5' end of the intron between exons 13 and 14 inside the rat ACE gene.

mRNA expression of ACE in kidneyRNA was isolated from kidney samples using the Nucleospin RNA kit (Macherey-Nagel) according

to the manufacturer’s protocol. Samples were diluted and 2µg was used for cDNA synthesis using the

Omniscript RT kit (Qiagen), with random hexamere primers. ACE mRNA levels were determined in

kidney, using a custom designed primer-probe set (Applied Biosystems) using the primers 5'-

CACCGGCAAGGTCTGCTT-3', 5'-CTTGGCATAGTTTCGTGAGGAA-3', and the probe 6-FAM

5'CAACAAGACTGCCA CCTGCTGGTCC-3'TAMRA. The ABI Prism 7900 HT sequence detection

system (Applied Biosystems) was used, which uses TaqMan chemistry for highly accurate quantitation

of mRNA levels. Renal ACE levels were expressed relative to those of the beta-2 microglobuline

housekeeping gene (B2M), because with geNorm VBA applet for Microsoft Excel (Vandensompele,

2002), B2M was determined as being the most stable housekeeping gene compared to the genes

encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucuronidase beta (GUSB). The

B2M probe and primers Rn00560865_mi were provided as preoptimized control system (Applied

Biosystems).

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ACE activityACE activity was determined as described previously [13]. Renal cortex tissue was homogenized

in a 50 mM K2PO4 buffer at pH 7.5. Subsequently, 100 µl of the diluted sample was pipetted into a 0.5

M K2PO4 buffer. Then, the substrate (100 µl of 12.5 mM Hip-His-Leu (Sigma)) which is cleaved by

ACE, was added. This was incubated at 37oC for exactly 15 minutes. In this amount the substrate is

present in excess, and thus not rate-limiting for the reaction. The conversion of the substrate was

stopped by adding 1.45 ml of 280 mM sodiumhydroxide. Then, 100 µl of 1% phtaldialdehyde, which

adheres to the formed bipeptid His-Leu, was added. The amount of tagged His-Leu was fluorimetrically

determined at 364 nm excitation wavelength and 486 nm emission wavelengths. This yields a measure

of the amount of His-Leu generated in the sample. In blank samples, sodiumhydroxide was added to

prevent conversion. The substrate was added after the incubation period. The coefficient of variation

was 6% for these measurements of ACE activity using this method.

ImmunohistochemistryACE immunohistochemistry was performed on frozen sections. Endogenous peroxidase was

blocked with 0.075% H2O2 in phosphate buffered saline (PBS, pH 7.4) for 30 minutes. Primary

antibodies (Chemicon International, CD143, MAB4051, clone 9B9) were incubated for 60 minutes at

room temperature. Binding was detected using sequential incubation with peroxidase-labeled

secondary and tertiary antibodies (Dakopatts, Glostrup, Denmark) for 30 min. All antibody dilutions

were made in PBS supplemented with 1% BSA and 1% normal rat serum was added to the secondary

and tertiary antibodies. Peroxidase activity was developed by using 3,3’-diaminobenzidine tetrachloride

for 10 min containing 0.03% H2O2. Counterstaining was performed using Mayer’s hematoxylin.

Contraction measurements in the isolated aortaImmediately after removal, the aorta was placed in a Krebs bicarbonate solution equilibrated with

95% O2 and 5% CO2 [14]. After the blood vessel was cleaned of connective tissue, eight rings of 2-

mm length were cut with a sharp razor blade, and care was taken not to touch the luminal surface. The

rings were mounted between two stirrups in organ baths filled with 15 ml Krebs solution (37.5°C) and

studied in parallel. One stirrup was anchored inside the organ bath, and the other connected to a

displacement transducer to determine isotonic changes [15]. Rings were subjected to 14 mN and

allowed to stabilize for 60 min, during which regular washing was performed. Rings were primed by

evoking a contraction with 10 µM phenylephrine (PE). Then a second response with 10 µM PE was

evoked, which served as a control contraction. These contractions were not significantly different

between the two rings studied in parallel or between different groups (data not shown). After washout

and renewed stabilization, rings were incubated with 100 µM NG-mono-methyl-L-arginine (L-NMMA)

for 45 min. Then, with L-NMMA still being present, parallel rings were stimulated with either

angiotensin I or angiotensin II (0.1 nM-1 µM bath concentrations). Finally, ang I induced response was

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studied in the presence of lisinopril. These rings were pre-incubated with lisinopril in addition to L-

NMMA. All results are expressed as percentage change of the maximal ang II induced response for

every pair of rings.

Statistical analysesTo avoid non-specific differences between rats (inter-assay differences), we evaluated the

responses to ang I in comparison to concentration-response curves to ang II obtained with parallel

rings of the same rat. To control for non-specific differences between rings from the same rat (intra-

assay variance), contractile responses to ang I and ang II were first normalized by calculating the

response as a percentage of the (reference) control contraction response to phenyllephrine (%PE). In

order to estimate the area between the concentration response curves of ang I and ang II, the area

under the curve was determined from both separately (Prism).

Subsequently, the difference between the areas under the curve was calculated. The same method

was used to estimate the area between the concentration response curves to ang I in the presence of

lisinopril. Data are expressed as median and interquartile range or mean and SEM for the response

curves. They were analyzed using the statistical program SPSS 12.0.2 for Windows and Graph Pad,

Prism, Version 4.03. Statistical differences were determined using Mann-Whitney U test. Significance

was accepted at p<0.05.

Results

Rat characteristics Body weight and urinary protein excretion are shown in table 1. Body weight was similar in BB and

LL, both at week 6 (time point used for aortic rings) and week 12 (time point used for ACE activity/

mRNA/24h urine collection). Urinary protein excretion was neglectable low in both groups.

ACE activity and mRNA expression of ACE ACE activity and ACE mRNA expression are presented in figure 1. Expression is shown as the

number of ACE mRNA molecules per molecule of B2M mRNA. ACE activity is approximately two times

higher in the BB in serum, heart and kidney homogenates (all, p<0.05, panel A-C, left side). In the

aorta homogenate, ACE activity is approximately two times higher in LL (p<0.05, panel D). Relative

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Wistar BB Wistar LL

Body weight (gram) week 6 122 (120-145) 92 (89-171)

week 12 375 (367-392) 354 (340-377)

Urinary protein (mg/24h) week 12 20 (17-26) 19 (15-21)

Table 1 │ Clinical characteristics of Wistar rats

Median and quartile range is presented

ACE mRNA expression is higher in kidney tissue (panel B, right side, *p<0.05) and non-significantly

higher in cardiac tissue (p=0.06, panel C, right side).

ACE immunohistochemistryIn the kidney, expression of ACE protein is present in glomerular visceral and parietal epithelial

cells, in the vascular endothelium of the large arteries, in the proximal- and distal -tubules (figure 2 A-

F). In BB there is a higher kidney (in all separate parts) ACE protein expression compared to LL, with

the most remarkable difference in tubular ACE protein expression. In heart tissue there was ACE

protein expression in the vascular endothelial cells (figure 2 G/H), without differences between BB and

LL. In LL aortic endothelial staining for ACE was positive. However, in aortic tissue from BB no

endothelial ACE staining was observed in aortic tissue (figure 2 I/J).

Vascular responsesThe contractile responses to equimolar concentrations of ang I and ang II in isolated parallel aortic

rings are presented in figure 3. Panel A and B show the responses to ang I, ang II and ang I + lisinopril

separately for BB and LL. It shows that ang I and ang II induced concentration-dependent

vasoconstriction in both strains. It also shows that difference (area) between the concentration

response curve to ang I and ang II was significantly higher in LL (panel C, p<0.05), indicating that there

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Figure 1 | ACE activity and relative ACE expression in Wistar BB vs LL. Expression is shown as the number of ACE mRNA molecules per molecule of B2M mRNA. The Wistar BB has higher ACE activity in serum, heart in kidney (panel A, B, C left side, # p<0.05). Relative ACE mRNA expression is higher in kidney

tissue from Wistar BB (panel B, right side, # p<0.05) and borderline significantly higher in heart tissue from Wistar BB (panel C, right side, p=0.06). In the aorta, ACE activity is in higher in Wistar LL (panel D, # p<0.05).

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Figure 2 | ACE staining in kidney, heart and aortic tissue

A

I

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FE

DC

B

J

was more conversion of ang I in the BB. The effect of lisinopril on the contractile response to ang I was

studied to assess the contribution of ACE in the conversion of ang I to ang II. Lisinopril induced a

significant shift to the right in the curves, demonstrating annihilation of the differences between the

strains in conversion from ang I to ang II (panel D, p= ns).

DiscussionIn this study we evaluated the functional and biochemical characteristics related to the ACE B/L

allele in the outbred Wistar rat. Serum ACE activity was higher is BB rats. Heart and kidney ACE

activity and mRNA expression were twofold higher in rats homozygous for the B allele and vascular

ang I conversion was enhanced in the Wistar BB rat. This enhanced conversion was reversible by

ACEi. These results are in line with data obtained in the human ACE I/D genotype. Therefore, the B

and L Wistar lines may provide a useful model to study the functional aspects of the human ACE

genotype.

Genetically, deletion in intron 16 of the human ACE gene results in an increased ACE activity both

on plasma and tissue level [1;16;17]. Numerous studies have been performed which related the ACE I/

D genotype to cardiovascular and renal disease [3;18;19]. Whereas meta-analyses confirm a role for

the D-allele as a renal and cardiovascular risk factor [20;21], yet many discrepancies are reported in

the literature [3]. A possible explanation for such discrepancies is the presence of multiple interactions

with both genetic, and environmental factors, i.e. sodium status, disease duration and gender. Animal

models would be helpful to unravel these complex interactions in well-controlled experimental

conditions. Therefore, we investigated whether the previously described difference in the B/L allele

between the Brown Norway and Lewis rats could be identified in the outbred Wistar rat. Secondly, we

bred a Wistar BB and LL line and determined its phenotype related to ACE, in terms of serum and

tissue ACE activity and expression in kidney and heart. Both on tissue and plasma level clear

differences in ACE activity and mRNA expression were found between the strains.

The levels of ACE activity in the serum, kidney and heart are in accord with earlier reports in

healthy Wistar rats [11;22-24]. The BB has approximately twice as high levels of ACE activity and

mRNA expression in serum, heart and kidney. This is in agreement with the human plasma and tissue

variation in relation to the ACE I/D genotype [1;16]. In order to provide a functional assessment of

differences in ACE activity on tissue ang I conversion, we included measurements of contractile

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Legend to figure 2Upper panels show kidney tissue of Wistar rat BB (A, close up C) vs LL (B, close up D) showing more staining of ACE in the tubules of the BB rat. Panel E and F show kidney vessels of BB (E) and LL (F), with more staining in BB. Panels G and

H show two coronary vessels with clear endothelial staining without differences between BB (G) and LL (H). Lower panels shows aortic tissue, without staining in BB (I) and endothelium staining in the LL rat (J).

responses to ang I and ang II in isolated aortic rings. These results show a consistent higher

conversion of ang I in the BB rat, which was reversible by lisinopril. This gives proof of a functional

higher level of ACE in BB, which can be annihilated by ACEi.

Our experimental setup in isolated aortic rings was similar to earlier studies that evaluated the

efficacy of ACEi from the difference in the dose-response curves for ang I and ang II as a functional

index of ACEi [13;25;26]. In comparison to the measurement of ACE activity in tissue homogenates,

this setup has the advantage that the normal vascular architecture is preserved. This is physiologically

relevant since the conversion of ang I then occurs at the site of action [27]. However, this setup has the

limitation that responses to exogenously administrated ang I and ang II may be similar [13], most likely

due to high ang I converting capacity in rat aorta. Therefore, the clear difference in ang I responses,

with higher responses in Wistar BB points towards a functionally relevant difference of the B/L allele.

These differences are annihilated during pre incubation with lisinopril.

In addition to the functional assessment of ACE activity in aortic rings, we also assessed ACE

activity in aortic homogenates, as cleavage of the substrate Hippuryl-His-Leu and performed

immunohistochemistry on aortic tissue. Interestingly, ACE activity was higher in Wistar LL and the BB

rat showed no staining of ACE in the aortic endothelium. The biochemical and staining results did not

parallel the functional findings. This may have various explanations. First, with respect to the

vasoconstrictor response to ang I, involvement of non-ACE dependent, like chymase ang II forming

pathways cannot be excluded. However, it should be noted that non-ACE pathways are under debate

in rats and that chymase can cause breakdown of ang II in rats [28]. The differences between ang I

responsiveness were not present during pre incubation with lisinopril. This raises the hypothesis that

another isoform of ACE could be present in the aorta, which is inhibited by lisinopril as well but does

not cleave Hippuryl-His-Leu and does not cross react with our immunohistochemistry antibody. As

noted above, in homogenates, in contrast to functional measurement in the aortic rings, the normal

vascular architecture is destroyed, which may affect the conversion of the substrate in various ways

that do not reflect the physiological condition. Finally, the ACE activity assay measures the conversion

of Hippuryl-His-leu to His-Leu. Other substances than ACE which cleave this product similarly may

have affected these measurements. But this doesn’t explain the discrepancy with the

immunohistochemistry data, which showed absence of ACE staining in Wistar BB. Which of the above

mechanisms might be involved in the discrepancies between functional ACE activity, biochemical ACE

activity and immunohistochemistry data; can not be derived from our data. However, these

discrepancies might be very relevant and reflect tissue specific regulation of ACE and need further

research.

We did not measure blood pressure. However, several studies in other rat strains with genetic

difference in ACE; healthy congenic, transgenic and Brown Norway vs Lewis rats have shown similar

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blood pressure levels [8;10;29;30]. Several association studies in humans using the ACE I/D genotype

did not show any significant relationship with blood pressure [31;32]. Therefore, it is not very likely that

there would have been a difference between the Wistar BB and LL. We have previously shown in the

Wistar rat that phenotypic individual differences in renal ACE activity predict the severity of

adriamycine-induced renal damage [11]. These result support the assumption that genetic differences

in (tissue) ACE activity predispose to a less favorable course of renal damage. Moreover, in response

to disease ACE is upregulated [33-35]. It would be very interesting in this respect to investigate

whether this up regulation would be influenced by the genetic background.

To identify the physiological mechanisms explaining the genetic modulation of serum ACE activity,

we measured the mRNA content of the kidneys. These measurements show clearly both the ACE

activity and mRNA expression were related to the B/L alleles and suggested that the ACE gene

expression is controlled at the transcriptional level. Challeh et al. showed previously that the ACE

transcription rate was nearly two-fold higher homozygote B rats compared to L rats [8]. The molecular

mechanism, explaining the difference in ACE activity related to the human ACE I/D genotype remain

unknown as well. The ACE I/D genotype is located in a non-coding region (intron 16). Several

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Figure 3 | Concentration-response relationship for ang I and ang II in Wistar BB (panel A) vs LL (panel B). Two parallel rings were stimulated with either ang I or II and studied for contractile responses. The results are expressed as a percentage of maximal response to ang II for every pair of rings (mean ± SEM). The area under the curves was

determined for the individual pairs and compared; Wistar LL had a significant larger area, reflecting lower conversion from ang I to ang II (panel C, *p<0.05). The effect of lisinopril (ACEi) on the contractile response to ang I was studied, which showed a shift towards the right in the curves, with subsequent no differences between the conversion from ang I to ang II

anymore (panel D).

possibilities have been forwarded. First, intronic variations can influence various regulatory

mechanisms of transcription, including splicing efficiency [36]. Thus, ACE I/D genotype could exert

effects on transcription via alteration of an undocumented regulatory pathway. Second, ACE I/D

genotype may be linked to another, functional polymorphism that is responsible for the differences in

ACE activity. Detailed haplotype and linkage disequilibrium studies have been performed resulting in

several candidate haplotypes [37], yet no conclusive evidence has been provided to date. It would be

very interesting to unravel the molecular mechanism behind difference in ACE expression in both

human and rats, this would greatly improve the understanding of ACE and its regulation.

Previous studies related pathophysiological differences between Brown Norway and Lewis strain

to variation in ACE B/L and ACE activity. However, a serious limitation to the approach of comparing

two different strains is the different genetic background where other genes can influence the

phenotype. The best solution would be to study the effect of the ACE gene in a congenic model.

However, producing congenic strains is technical difficult and time consuming. Only one article with

congenic rats has been published. In a diabetic model, increased TGF-B1 expression, in the rats with

higher ACE expression was found [29]. The Wistar rat seems a good alternative for a congenic strain,

as only selecting for the ACE genotype while keeping the rest of the genetic background random

cancels the effect of other genes on the phenotype in the two different lines. We showed a functional

difference in ang I conversion, which Challah et al. did not show previously when comparing the Brown

Norway and Lewis strain [8]. This is in line with the functional effect of the ACE I/D genotype in which

enhanced ang I conversion has been shown but not uniformly [5;38;39]. Our results are comparable to

those of a transgenic over expression model [30]. In an isolated heart set up, coronary flow response

to ang I was increased in the transgenic hearts compared with the control group with increasing

dosages. This effect was reversible upon a co-infusion with an ACEi.

In conclusion, the B/L allele variation in the ACE gene of the Wistar rat may provide a good model

for the human ACE I/D genotype with higher plasma and tissue ACE levels in Wistar BB. Moreover,

functional conversion of ang I is enhanced in Wistar BB, which is reversible upon ACEi. We can not

explain the differences between the functional measurement and ACE activity / immunohistochemistry

data in the aortic wall. These data emphasize the importance of further studies trying to explain how

the ACE gene polymorphism interact with cardiovascular pathologies and define more exactly its

molecular basis in animal studies as well as in humans.

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