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LacZ mice, an animal model for the autosomal dominant polycystic kidney disease type 2 (ADPKD2)
International Journal of Cardiology 120 (2007) 158 – 166 www.elsevier.com/locate/ijcard
Cardiovascular characterization of Pkd2 +/LacZ mice, an animal model for the autosomal dominant polycystic kidney disease type 2 (ADPKD2) Jörg Stypmann a,b,1 , Markus A. Engelen a,c,1 , Stefan Orwat a , Konstantinos Bilbilis d , Markus Rothenburger e , Lars Eckardt a , Wilhelm Haverkamp a,b , Jürgen Horst d , Bernd Dworniczak d , Petra Pennekamp d,⁎ b
a Department of Cardiology and Angiology, Hospital of the University of Münster, Germany Interdisciplinary Center for Clinical Research (IZKF), Central Project Group 4 (ZPG4a), Münster, Germany c Department of Medical Physiology, University Medical Center Utrecht, The Netherlands d Institut für Humangenetik, Universitätsklinikum Münster, Vesaliusweg 12, D - 48149 Münster, Germany e Department for Thoracic and Cardiovascular Surgery, Hospital of the University of Münster, Germany
Received 21 February 2006; received in revised form 27 August 2006; accepted 20 September 2006 Available online 19 December 2006
Abstract Background: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2. Patients with ADPKD have an increased incidence of cardiac valve abnormalities and left ventricular hypertrophy. Systematic analyses of cardiovascular involvement have so far been performed only on genetically unclassified patients or on ADPKD1 patients, but not on genetically defined ADPKD2 patients. Even existing Pkd1 or Pkd2 mouse models were not thoroughly analyzed in this respect. Therefore, the aim of this project was the noninvasive functional cardiovascular characterization of a mouse model for ADPKD2. Methods: Pkd2+/LacZ mice and wildtype controls were classified into 8 groups with respect to gender, age and genotype. In addition, two subgroups of female mice were analyzed for cardiac function before and during advanced pregnancy. Doppler-echocardiographic as well as histological studies were performed. Results: Doppler-echocardiography did not reveal significant cardiovascular changes. Heart rate and left ventricular (LV) length, LV mass, LV enddiastolic and LV endsystolic diameters did not differ significantly among the various groups when comparing wildtype and knockout mice. There were no significant differences except for a tendency towards higher maximal early and late flow velocities over the mitral valve in old wildtype mice. Conclusions: Non-invasive phenotyping using ultrasound did not reveal significant cardiovascular difference between adult Pkd2+/LacZ and WT mice. Due to the lack of an obvious renal phenotype in heterozygous mice, it is likely that in conventional ADPKD knock out mouse models severe cardiac problems appear too late to be identified during the reduced lifespan of the animals. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Polycystin-2; Pkd2; APKD; Cystic kidney disease; Ultrasound; Echocardiography
1. Introduction Autosomal dominant polycystic kidney disease (ADPKD) is a frequent hereditary renal disorder with an overall incidence of 1 to 2.5 in 1000 [1]. It is characterized by the ⁎ Corresponding author. Tel.: +49 251 8355405; fax: +49 251 8356995. E-mail address: [email protected] (P. Pennekamp). 1 The first two authors contributed equally to this work. 0167-5273/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2006.09.013
formation of multiple, fluid-filled cysts not only in both kidneys but also in other organs [2]. About 50% of PKD patients develop end-stage renal failure (ESRF) by late middle age [3], accounting for 5–10% of patients requiring haemodialysis world wide [4]. However, the course of the disease frequently is complicated by a multiplicity of extra renal manifestations: even in patients with normal renal function, nearly 70% suffer from hypertension; many patients develop left ventricular hypertrophy and 20% suffer from
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intracranial aneurysm. Furthermore, patients can be affected by cardiac valvular abnormalities, colonic diverticuli, and inguinal hernia. In the meantime, ADPKD is regarded as a multisystemic disorder [5]. So far, two genes have been identified which are mutated in the majority (99%) of ADPKD cases: PKD1, encoding polycystin-1 (PC1), an integral membrane-spanning glycoprotein suggested to function as epithelial cell membrane mechanoreceptor, sensing morphogenetic cues in the extracellular environment, and PKD2, encoding polycystin-2 (PC2), a membrane-spanning protein as well functioning as a Ca2+ permeable cation channel [6–8]. Both polycystins are likely to function in a common pathway because mutations in either gene cause strikingly similar phenotypes. Although the disease displays inter- and intrafamilial phenotypic heterogeneity [9], detailed studies revealed that in the long run ADPKD has a milder course in families linked to PKD2. These patients suffer later from symptoms, exhibit a smaller number of cysts at the time of diagnosis, and chronic renal failure threaten PKD2 patients later in life as compared to PKD1 patients [10]. Orthologues of the human PKD1 and PKD2 genes exist in the mouse genome, and several different knockout mice have been created. Heterozygous mice appear normal but develop single cysts in the kidney or liver late in life and have a reduced overall lifespan [11]. Homozygous null mutant mice die in utero with massively enlarged cystic kidneys, pancreatic ductal cysts and pulmonary hypoplasia and often exhibit edema, vascular leaks, and rupture of blood vessels suggesting that the polycystins are required for the structural integrity of blood vessels [12,13]. In addition most of the homozygous embryos display multiple cardiac abnormalities including cardiac septation defects, double outlet right ventricle and pericardial effusions [11,14,15]. Severe cardiovascular abnormalities in homozygous mutant PKD1 or PKD2 knockout mouse embryos might be due to loss of polycystin function in the heart tissue which normally shows a high expression of both genes (Figs. 2 and 3; [14]). Moreover, patients frequently develop left ventricular hypertrophy before developing hypertension [16]. Therefore, the cardiovascular complications in ADPKD patients begun to be recognized as a primary defect due to the loss of PC1 and/or PC2 function in cardiovascular organs and not solely as a secondary defect due to decreasing renal function. However, owing to the similarity of the overall clinical phenotypes of ADPKD1 and 2 patients, it is assumed that the cardiovascular phenotypes in ADPKD1 and ADPKD 2 patients are virtually identical [17]. But, systematic analyses of the cardiovascular involvement have so far been performed only on genetically unclassified patients or on ADPKD1 patients, but not on genetically well defined panels of ADPKD2 patients. Even existing Pkd1 or Pkd2 mouse models were not thoroughly analyzed in that respect. Therefore, in the present study the cardiovascular performance of the Pkd2+/LacZ mouse as a mouse model for the ADPKD2 was investigated in more detail using noninvasive Doppler-
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echocardiography of heterozygous and wildtype mice of different gender and age. In addition, two subgroups of mice before and during advanced pregnancy were examined to clarify whether pregnancy deteriorates the heart performance in this mouse model of ADPKD2. 2. Methods 2.1. Generation and maintenance of Pkd2-deficient mice Pkd2-deficient mice have been generated by gene targeting in mouse embryonic stem cells as described previously [15]. In this mouse line, we replaced Pkd2 exon 1 and part of intron 1 by LacZ as a reporter gene. LacZ was fused to the original Pkd2 ATG start codon enabling to monitor LacZ expression under the control of the Pkd2 promotor (Mouse Genome Informatics: allele Pkd2tm1Dwo (http://www.informatics.jax. org). In this report, heterozygous Pkd2 mutated mice are named Pkd2+/LacZ and homozygous Pkd2 mutated mice are named Pkd2LacZ/LacZ). For the present study, Pkd2+/LacZ mice on a mixed C57/BL6 and 129/Sv genetic background were used. Mice were conventionally housed in a controlled environment at a temperature of 21 °C ± 1 and a 12 h day/night cycle. Mice were kept in macrolon cages and had access to standard food pellets and tap water ad libidum. All experimental procedures had been approved by the local government authorities and confirm to the NIH Guide for the Care and Use of Laboratory Animals. 2.2. Expression analysis by LacZ staining Whole embryos were fixed in 4% PFA/PBS + 2 mM MgCl2 for 5–30 min at room temperature and washed 3 times in 1 × PBS, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40. LacZ staining was performed by incubation of samples with 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 3H2O, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, and 1 mg/ml X-Gal (4-chloro-5-bromo-3-indolyl-β-Dgalactopyranoside) in 1 × PBS (pH 7.4–8.0) at 30 °C. After staining, samples were washed in 1 × PBS, postfixed in 4% PFA, dehydrated in methanol, and embedded in Historesin (Leica Microsystems, Bensheim, Germany). Stained embryos were sectioned at 4 μm on a MICROM HM 355 microtome (MICROM, Walldorf, Germany) and staining was visualized under darkfield illumination by use of a Zeiss Axioskop microscope. Adjacent sections were counterstained with methylen blue/azur II and visualized under bright field illumination. Adult organs were frozen and sectioned on a MICROM HM 500 O microtome (MICROM, Walldorf, Germany) at 20 μm for X-Gal staining as described above and counterstained with eosin. 2.3. Grouping of mice for analysis Pkd2+/LacZ mice and their wildtype littermates were classified into 10 groups with respect to gender, age and genotype as
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Table 1 Basic characteristics of all Doppler-echocardiographically examined mice Group
I
Age
102 ± 33 days
II
Gender
Female
Genotype Number of animals Body weight [g] Heart rate [bpm]
Pkd2+/LacZ 21 22.1 335 ± 58
Ib
IIb
III
IV
V
VI
VII
VIII
483 ± 69 days
WT 23 22.3 322 ± 70
Female/pregnant
Male
Pkd2+/LacZ 7 33.79 387 ± 145
Pkd2+/LacZ 6 28.6 333 ± 74
WT 5 33.04 356 ± 38
shown in Tables 1 and 2. In addition, after the first examination several female mice out of groups I and II were mated to analyze cardiac performance during pregnancy between embryonic day (E) 15 and E17 (subgroups Ib and IIb). The age of the young animals (groups I–IV) varied between 2 and 5 months (mean age 102 ± 33 days), the age of the old animals (groups V–VIII) varied between 14 and 16 months (mean age 483 ± 69 days). 2.4. Doppler-echocardiography Doppler-echocardiographic examination was performed as previously described in detail [18–20]. The investigator
Female WT 11 30.0 353 ± 70
Male
Pkd2+/LacZ 6 38.3 346 ± 33
WT 6 38.6 312 ± 95
Pkd2+/LacZ 6 40.9 357 ± 48
WT 6 41.0 323 ± 46
was blinded with regard to the genotype of the mice. After induction of light anesthesia with a combination of 50 mg/kg ketamin (Ketavet®, Pfizer Pharma GmbH, Karlsruhe, Germany) and 5 mg/kg xylazine (Rompun®, BayerVital, Leverkusen, Germany), the chests of the mice were precordially shaved. The mice were secured in a supine position to a warm waterbed heated to 38 °C and angled 45° to the left. One lead ECG was used to monitor heart rate. Transthoracic echocardiography was performed using a commercially available digital cardiac ultrasound platform equipped with either a 6- to 12-MHz short focal length-phased array transducer equipped with an acoustic stand-off or a 15-MHz linear-array transducer (Sonos 5500, B1-software package,
Table 2 Doppler-echocardigraphic data of left ventricular performance with subgroup analyses Group
I
Age
102 ± 33 days
II
Gender
Female +/LacZ
Female/pregnant
Pkd2 1.99 ± 0.31 0.82 ± 0.07 4.29 ± 0.91 2.76 ± 0.71 0.88 ± 0.09 5.97 ± 0.45 35.08± 6.80
WT 1.96 ± 0.38 0.85 ± 0.11 4.28 ± 1.13 2.68 ± 0.68 0.94 ± 0.10 6.00 ± 0.76 37.04 ± 5.96
LV-EF [%] Mass of LV [mg] LV mass index [mg/g] LV-CO [ml/min] Cardiac index [ml/min/g] Ao Vmax [cm/s] Ao PGmax [mmHg] Ao MPG [mmHg]
57 ± 9 92 ± 25 4.28 ± 1.32 10 ± 2 0.47 ± 0.13 59.7 ± 6.6 1.46 ± 0.33 0.779 ± 0.184 42.7 ± 8.8 21.3 ± 5.2
60 ± 8 100 ± 33 4.50 ± 1.63 11 ± 3 0.50 ± 0.16 60.8 ± 6.0 1.49 ± 0.30 0.793 ± 0.176 47.2 ± 10.3 23.6 ± 8.0
2.08 ± 0.53 0.271 ± 0.093
2.12 ± 0.57 0.300 ± 0.146
Ratio of E/A-Wave MV PGmean [mmHg]
IIb
III
IV
V
VI
VII
VIII
483 ± 69 days
Genotype LA [mm] LVAWd [mm] LVEDd [mm] LVESd [mm] LVPWd [mm] LV-length [mm] LV-FS [%]
MV E-wave [cm/s] MV A-wave [cm/s]
Ib
+/LacZ
Pkd2 2.14 ± 0.33 0.88 ± 0.04 4.00 ± 0.83 2.35 ± 0.65 0.66 ± 0.05 6.67 ± 0.66 42.05± 4.56 66 ± 6 100 ± 19 2.98 ± 0.51 18 ± 4 0.53 ± 0.13 68.30 ± 7.2 1.89 ± 0.40 0.96 ± 0.750 48.14 ± 6.8 30.46 ± 16.6 1.93 ± 0.68 0.33 ± 0.080
WT 2.02 ± 0.37 0.82 ± 0.06 3.75 ± 0.63 2.20 ± 0.31 0.64 ± 0.07 6.53 ± 0.48 40.67 ± 5.97 64 ± 7 91 ± 9 2.77 ± 0.31 17 ± 1 0.53 ± 0.03 68.94 ± 7.2 1.93 ± 0.40 0.75 ± 0.340 55.94 ± 6.5 29.04 ± 7.76 2.01 ± 0.33 0.40 ± 0.140
Male +/LacZ
Pkd2 1.90 ± 0.14 0.77 ± 0.08 3.43 ± 0.29 1.89 ± 0.31 0.84 ± 0.10 6.46 ± 0.79 45.04 ± 6.75 69 ± 8 74 ± 13 2.60 ± 0.34 14 ± 3 0.49 ± 0.12 77.1 ± 13.5 2.44 ± 0.82 1.239 ± 0.390 58.7 ± 11.8 25.8 ± 5.5
Female WT 2.02 ± 0.30 0.79 ± 0.09 4.00 ± 0.85 2.36 ± 0.63 0.86 ± 0.11 6.57 ± 0.28 41.50 ± 6.79 65 ± 8 90 ± 19 2.99 ± 0.55 15 ± 4 0.50 ± 0.14 75.0 ± 8.9 2.28 ± 0.57 1.230 ± 0.315 62.8 ± 14.1 30.7 ± 8.9
+/LacZ
Pkd2 1.84 ± 0.29 0.91 ± 0.10 2.85 ± 0.25b 1.92 ± 0.18b 1.05 ± 0.14b 6.01 ± 0.45 32.39 ± 5.88 54 ± 8 78 ± 17 1.97 ± 0.38b 17 ± 6b 0.42 ± 0.10 58.6 ± 5.8 1.39 ± 0.28 0.736 ± 0.180 41.1 ± 4.4 20.6 ± 3.5
2.33 ± 0.51 2.09 ± 0.35 2.05 ± 0.47 0.372 ± 0.472 ± 0.295 ± 0.078 0.147 0.123
Male WT 1.80 ± 0.09 0.94 ± 0.11 3.05 ± 0.14 2.08 ± 0.04 1.02 ± 0.08a 5.90 ± 0.63 31.72 ± 2.03 53 ± 3 81 ± 14 2.12 ± 0.39c 14 ± 3 0.35 ± 0.08 62.0 ± 3.8 1.54 ± 0.19 0.837 ± 0.097 39.7 ± 7.6 20.8 ± 4.9
Pkd2+/LacZ 2.00 ± 0.39 0.94 ± 0.06d 3.10 ± 0.26 1.90 ± 0.15 1.02 ± 0.07 6.27 ± 0.61 38.38 ± 4.57 62 ± 6 86 ± 8 2.14 ± 0.37 16 ± 2 0.41 ± 0.13 63.6 ± 1.6 1.62 ± 0.09 0.841 ± 0.076 54.5 ± 13.3 18.1 ± 6.3
WT 2.12 ± 0.22 1.01 ± 0.13e 3.26 ± 0.34 1.84 ± 0.21 0.97 ± 0.11 6.09 ± 0.61 43.38 ± 4.39 68 ± 5 91 ± 22 2.42 ± 0.78 16 ± 3 0.43 ± 0.14 75.8 ± 9.1 2.33 ± 0.53 1.123 ± 0.285 57.3 ± 4.5 24.0 ± 3.8
1.95 ± 0.28 0.215 ± 0.070
3.21 ± 1.09 0.375 ± 0.214
2.43 ± 0.35 0.364 ± 0.088
Ao PGmax: aortic maximum pressure gradient [mmHg]; Ao MPG: mean aortic pressure gradient [mmHg]; Ao Vmax: aorta velocity maximum [cm/s]; LVAWD: LV anterior wall enddiastolic diameter; LVPWd: LV posterior wall enddiastolic diameter; LA: left atrial diameter; LV: left ventricle; LVEDD: left ventricular enddiastolic diameter; LV-EF: LV-ejection fraction; LVESD: left ventricular endsystolic diameter; LV-FS: LV-fractional shortening; LVOT-CO: Dopplerechocardiographic cardiac output via left ventricular outflow tract; MV A-Wave: atrial inflow of mitral valve; MV E-Wave: early inflow of mitral valve; MV PGmean: Mean pressure gradient of mitral valve. Significances p b 0.05: a = V/VI, b = I/V, c = II/VI, d = III/VII, e = IV/VIII.
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Fig. 1. Representative echocardiographic images. Parasternal long axis view (A), M-mode (B), Doppler flow signals of the mitral inflow with E- and A-wave (C) and Doppler flow over the aortic valve (D). Velocity of the Doppler flow signals shown on the right side of the images are given in m/s. Exemplary image of an aortic insufficiency (E). LV = left ventricle, LA = left atrium, Ao = aorta.
Philips, The Netherlands). A smooth layer of 1.5–2 cm thick centrifuged ultrasound gel was placed on the chest of the mice and the probe was gently dipped into this layer of gel avoiding any pressure on the thorax to prevent thorax deformation or reflex bradycardia. Parasternal long-axis and short-axis views were obtained at a frame rate of 300 per second. The variable depth of the zoom box was set at 2– 4 cm. M-mode and Doppler recordings were performed at a sweep speed of 150 mm/s. M-Mode data of the LV anterior (LVAWd) and posterior wall thickness (LVPWd) at enddiastole as well as end-diastolic and end-systolic dimensions of the left ventricle were acquired in the parasternal long axis view following the leading edge to leading edge method [20] of three consecutive cycles. Percentage of fractional shortening (FS) as an index of LV systolic function was calculated using the equation [18,20–22] FSð%Þ ¼
LVEDD−LVESD d100: LVEDD
Echocardiographic LV mass was calculated using the arealength method as previously described [22–24] and corrected for bodyweight by calculating the LV mass index [18]. Pulsed wave Doppler signals were obtained in a modified apical view by placing the sample volume parallel to the flow into LV outflow tract (LVOT) for systolic outflow of the left ventricle as well as apical to the mitral valve within left ventricle for diastolic inflow. The left atrium was checked for mitral regurgitation by pulsed wave Doppler. In addition, mitral and aortic valves were examined for regurgitation by color flow Doppler. Cardiac output was calculated using
heart rate, velocity time integral of the Doppler-signals in the LV outflow tract and the end-diastolic diameter of the LVOT [21]. 3. Histological analyses For light microscopy, organs were fixed in 4% paraformaldehyde dissolved in 1 × PBS (4% PFA/PBS) solution and embedded in paraffin. Sections of 6 μm were stained with methylen blue/azur II and analyzed by light microscopy (Axioskop, Zeiss, Germany). 4. Statistical analyses Values were calculated with SPSS 11 statistical software and expressed as means and their respective standard errors or standard deviations. Groups I/II, I/Ib, II/IIb, Ib/IIb, III/IV, V/VI, VII/VIII, I/V, II/VI, III/VII and IV/VIII were compared using the two-tailed Student's t test and the Wilcoxon rank sum test. A p b 0.05 was considered to indicate statistically significant differences between the groups investigated. 5. Results 85 mice were included in this study including 12 female mice which were examined before and during pregnancy. As expected, the body weight of female non-pregnant mice (25.9 ± 8.4 g) was significant lower than the body weight of male mice (33.8 ± 6.8 g) (p b 0.0001) and body weight increased with age in both genders. No significant differences of body weight or heart rate have been observed between
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Fig. 2. Pkd2 expression in the heart of adult Pkd2+/LacZ mice. Pkd2 expression is reduced in the cardiac region of adult mice, but still can be detected in the cardiac valves and the septum membranaceum as demonstrated by X-Gal histochemistry (blue dots in A I, A II, B I and B II). Frontal sections of a heart isolated from a Pkd2+/LacZ mouse (A and B) and higher magnifications of the aortic valve (A I), tricuspid valve (A II), mitral valve (B I) and septum membranaceum (B II) viewed under bright field illumination.
Fig. 3. Pkd2 expression and abnormal cardiovascular development in Pkd2LacZ/LacZ embryos and a representative kidney section of a male Pkd2+/LacZ mouse at the age of 16 month. In this mouse line, LacZ is fused to the Pkd2 ATG start codon enabling to monitor LacZ expression under the control of the Pkd2 promotor using whole mount X-Gal staining. Viewed under darkfield illumination, blue X-Gal precipitates appear pink. Expression to different extend was detectable in all structures of the heart with the highest expression in the outflow tracts, especially the endocardial cushions and valve leaflets, and lower levels in the myocardium. Mutant embryos showed cardiovascular abnormalities to different extend including disorganization of the myocardial wall and ventricular septum, dysplastic endocardial cushions, double outlet right ventricle (DORV) and single chambered ventricle. Frontal (A–F) and sagital sections of WT (A), Pkd2+/LacZ (B, G) and Pkd2LacZ/LacZ (C–F, H) embryonic hearts and kidney section of a male Pkd2+/LacZ mouse at the age of 16 month (I). A, B: newborn; C, E– H: E16.5; D: E17.5; I: E15.5; A–F: X-Gal staining viewed under dark field illumination; G–I: bright field illumination; G, H, I: methylen blue/azur II staining.
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Pkd2+/LacZ and wildtype mice of the same age and gender (Table 1). Heart rate did not differ significantly between all groups. The anesthetic regimen did not produce significant, prolonged or temporally variable changes in heart rate during the examination. Adequate Doppler-echocardigraphic measurements could be obtained in all mice without any problems within less than 20 min (mean less than 15 min). Representative images are shown in Fig. 1. Dopplerechocardiographic measurements and calculated values are shown in Table 2. Overall, there were not much striking significant differences between the compared groups. Color flow Doppler revealed insufficiencies of the aortic valve in one young female Pkd2+/LacZ mouse (group I) and in one young male wildtype mouse (group IV). LVEDD and LVEDS were significantly decreased in the old female Pkd2+/LacZ mice as compared with young heterozygous knockout mice. LVPWd and CO increased significantly with age in the female Pkd2+/LacZ mice. LVAWd was significantly increased as well in the old male Pkd2+/LacZ mice as in the old wildtypes as compared with younger mice of the respective genotype and gender. Ao Vmax decreased significantly with age in the Pkd2+/LacZ mice. FS, Ewave and A-wave were higher in male mice independent of the genotype when compared to females of the same age. Both gender and genotypes showed a tendency to an increase in cardiac output at older ages, but this trend cleared out after correction for body-weight. The observed time pattern of sonomorphometric indices during pregnancy and changes in functional Doppler-echocardiographic measurements in the examined mice did not show significant differences between Pkd2+/LacZ and wildtype mice. In summary, there were no significant differences between age-matched Pkd2 heterozygous mice and wildtype controls. As previously shown, embryos lacking polycystin-2 (Pkd2LacZ/LacZ) exhibited severe defects in cardiac morphogenesis including ventricular septal defects (VSD), abnormal formation of cardiac valves and transposition of the great vessels (TGA) [15,11]. During mouse embryogenesis, polycystin-2 is, similar to polycystin-1 [14], highly expressed in the conotruncus, especially in the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. In adult animals, expression of polycystin2 is decreased in the cardiac region but still can be detected in the cardiac valves and the septum membranaceum (Fig. 2). In the kidney, only few cysts can be detected in adult heterozygous knockout mice older than one year (Fig. 3). As expected, renal function in these mice is not affected ([11] and unpublished own data). 6. Discussion In recent years, autosomal dominant polycystic kidney disease (ADPKD) became increasingly recognized as a systemic disorder and on the cellular level this seems to
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be reasonable due to the wide spread expression of both genes. Although ADPKD patients suffer from many extrarenal manifestations [2], cardiovascular problems are the most severe side effects and several studies have been conducted to characterize the cardiac involvement in ADPKD patients. Cardiovascular problems seem to be a continuous process starting early during the course of the disease and it has to be pointed out that they are still a major cause of morbidity and mortality in patients with ADPKD [25,26]. Hypertension and left ventricular hypertrophy is common and associated with faster progression to end stage renal disease [16,25,27]. Especially hypertension develops in the majority of patients before any decrease in renal function can be determined [27]. It has been proposed that formation and expansion of renal cysts activate the renin–angiotensin– aldosterone system (RAAS) by which development of hypertension is triggered [28]; consistent with this hypothesis activation of RAAS can be verified well before onset of hypertension or any other clinical finding in ADPKD patients [29,30]. Recent studies showed that patients benefit from lowering the mean arterial pressure and by use of angiotensin-converting enzyme inhibitors which significantly slow down the decrease of renal function [31]. Beside hypertension and left ventricular hypertrophy, the prevalence of mitral-valve prolapse, mitral regurgitation, aortic insufficiency, tricuspid incompetence, and tricuspidvalve prolapse is increased [32]. The same holds true for the left ventricular mass (LVM) index and it was clearly shown that left ventricular dysfunction increases if ADPKD patients become hypertensive [33]. The same study revealed that the majority of valvular abnormalities occurred in dialysis patients which were generally related to annular calcification of the mitral or aortic valve. However, despite the feasibility of subdividing ADPKD patients into genetically defined groups (ADPKD type I caused by mutations in PKD1 versus ADPKD type 2 caused by mutation in PKD2) up to now only a single study has been published comparing a genetically classified cohort of ADPKD type 1 patients to unaffected family members and healthy controls [34]. In addition, although Pkd1 and Pkd2 mouse models are available, there are to the best of our knowledge no published data concerning the question whether cardiac performance is impacted in these animals. But, embryos lacking polycystin-2 and some of the mouse mutants for polycystin-1 [11,14,15] exhibit severe defects in cardiac morphogenesis including ventricular septal defects (VSD), abnormal formation of cardiac valves and transposition of the great vessels (TGA). Furthermore, during mouse embryogenesis, polycystin-2 is highly expressed in the conotruncus, especially the endocardial cushions, structures that give rise to the affected heart valves and the membranous ventricular septum. Later in development, expression of the polycystins can still be detected in the cardiac valves and great vessels. The prominent cardiac expression patterns for both polycystins suggest a relevant function of
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both proteins not only during development but in addition for maintenance of cardiac valves during adulthood. These findings let us assume that Pkd knockout mice can serve as model to analyze cardiac involvement in ADPKD in more detail. As initial step we therefore performed a noninvasive analysis of the cardiac performance of heterozygous mice of different gender and age using Doppler-echocardiography. In addition, two subgroups of female mice were analyzed for cardiac function before and during advanced pregnancy. Our echocardiographic data correspond very well with previously published values obtained in similar analyses using C57/Bl6 mice [24,35]. Despite the assumed function of the polycystins during development of cardiac structure and during adulthood, we did not detect significant pathological cardiovascular changes neither in young nor in older Pkd2+/LacZ mice of either gender or during pregnancy. Particularly, there were hardly any differences in respect to wall thickness, LV mass and occurrence of valvular insufficiencies. The significant change in LV mass index of the young versus the old female mice is due to changes of the body-weight during aging. If Pkd2+/LacZ mice had developed arterial hypertension, we would expect significant differences in left ventricular mass due to different load, which was not the case. These surprising findings might be due to a couple of different reasons: The course of the disease of ADPKD1 or ADPKD2 patients is in general clinically hardly to distinguish, but long term investigations uncovered that disease progression is milder in ADPKD2. Exemplary, onset of end-stage renal disease is later and ADPKD2-patients are less likely to have hypertension, urinary-tract infection or hematuria [10] and the same might be true for cardiovascular problems. However, following the course of the disease in human patients, independent whether it is caused by PKD1 or PKD2 mutations, severity of most symptoms clearly progress with increasing age of affected patients. Assumed that progression of disease is comparable fast in humans and mice, this ageing effect is only mimicked partially in mice. Cyst formation in kidney should serve as an example: Even though the mutational mechanism underlying initiation of cyst formation in ADPKD remains controversial the most widely accepted hypothesis proposes a two-hit mechanism of cyst formation to explain the focal nature of cyst development. However, frequency of the second hit clearly depends on time and total number of renal cells susceptible for the loss [36–38]. Indeed, cyst formation in conventionally targeted Pkd1 and Pkd2 mice is only detected rarely and their number and relative size is reduced as compared to humans [11] and resulting reduction of the renal function in these mice is obviously too low to impact cardiac function. Nevertheless, approaching the age of 15–18 months, many Pkd2+/LacZ mice suddenly die ([11] and unpublished own observations). Macroscopic investigations of deceased animals – if possible – suggest that death was either caused by ruptures of blood vessels or of large liver cysts similar to what is described for humans [39,40,25,41,42].
However, we could not identify the reason for sudden death in all animals investigated. Nevertheless, data form us and other groups show that haploinsufficiency of either Pkd1 or Pkd2 is sufficient to reduce survival of mutant mice [11,43,44]. Interestingly, it has been shown that loss of Polycystin 1 or 2 function and haploinsufficiency of either Pkd1 or Pkd2 deteriorates the structural integrity of the blood vessel walls [13,43,44] suggesting that sudden ruptures of these vessels might be a major cause of sudden death in these animals. But, if decreased survival of mice for which we could not identify the cause of death would be caused by cardiac failure, cardiovascular abnormalities have to develop very rapid because in part of this study we examined a rather old mouse population (14–16 months old) short before animals reached the end of the average life span and did not find gross structural or functional changes explaining forthcoming sudden death. If cardiac involvement in ADPKD is not only secondary but due to loss of polycystin function in the heart tissue itself – as we hypothesize – again average life span of Pkd2+/LacZ mice seems to be too short to emerge. Thus, these changes are hardly to detect in commonly sized cohorts of mice. Even triggering the development of cardiovascular problems by additional stress – in our case we measured heart performance before and during pregnancy – did not uncover pathological findings. It is known that in human disease progression is influenced by the type of the germ line and somatic mutations and so far unknown modifier genes. Especially in animal models it could be shown that environmental factors e.g. diet and housing conditions, could also affect disease progression [45]. To exclude an influence of the environmental factors on disease progression, especially housing and diet were kept constant during the complete study. Data from the literature and own observations suggest that in mice modifier genes play an important role in disease progression. In case of Pkd2 this indeed results in a more severe phenotype in homozygous mutant embryos on a 129/Sv genetic background compared to a pure C57/BL6 genetic background (unpublished own observation) leading to an earlier prenatal death in 129/Sv embryos. In this study, we examined F1 and N2 mice derived from backcrosses between 129/Sv (donor strain) and C57/BL6 (recipient strain) with the F1 generation providing especially the mice of the 14–16 month old mouse population. Though we cannot exclude the influence of modifier genes on the cardiac performance of heterozygous Pkd2 mutant mice, at least in the genetically homogenous F1 generation, the influence of these modifier genes should have been minimized if not excluded. During the complete study environmental factors, especially housing and diet, were kept constant to eliminate any possible influence of these factors. This tightly controlled study enabled us to evaluate solely the influence of haploinsufficiency of the Pkd2 mutation on to cardiac performance and resulting data can be used as base line for future studies.
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7. Limitations One limitation of this study is the low medium heart rate due to the use of ketamin–xylazine–narcosis. Most available narcotics have an impact on cardiac function of the mouse [46,47], partly by their influence on autonomic tone, partly due to a direct cardiodepressive effect. The normal heart rate of mice lies around 475–540 bpm [48] but heart rate is clearly depressed by most anesthetic drugs. Anesthesia by inhalation of volatile anesthetics like isoflurane or sevoflurane has less impact on cardiac function and heart rate. Some groups have performed echo in awake mice [47,49,50], but this technique requires a lot of training of the animals, a second researcher and much more time. Furthermore, this technique causes a high level of stress for the mice which again changes cardiac function and especially will increase heart rate. Finally, a change of cardiac morphology by fixing the mouse in a supine position cannot be excluded. 8. Conclusion In conclusion, the cardiac performance of our mouse model for Pkd2 seems to be not affected by partial loss of polycystin 2 function. This diagnostic finding is likely due to the fact that the life span of mice is too short to develop cardiovascular problems. Although it could be of some interest to analyze the cardiovascular phenotype of the different Pkd1 knockout mouse models we do not expect tremendous differences compared to our result. Acknowledgement Parts of these data are part of the doctoral thesis of Stefan Orwat. We thank Axel Bohring for helpful discussions. This work was partly supported by the Interdisciplinary Center for Clinical Research (IZKF) Münster, project numbers ZPG4a and BD IKF 2B 4 and by grants from the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich 656 MoBil Münster, Germany (project C3). Recent address of Wilhem Haverkamp: Medizinische Klinik mit Schwerpunkt Kardiologie, Campus VirchowKlinikum Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D – 13353 Berlin, Germany. References [1] Gabow PA. Autosomal dominant polycystic kidney disease. N Engl J Med 1993;329:332–42. [2] Gabow PA. Autosomal dominant polycystic kidney disease—more than a renal disease. Am J Kidney Dis 1990;16:403–13. [3] Gabow PA, Johnson AM, Kaehny WD, et al. Factors affecting the progression of renal disease in autosomal-dominant polycystic kidney disease. Kidney Int 1992;41:1311–9. [4] Wilson PD. Polycystic kidney disease. N Engl J Med 2004;350: 151–64. [5] Perrone RD. Extrarenal manifestations of ADPKD. Kidney Int 1997;51:2022–36.
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[6] Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. The international polycystic kidney disease consortium, vol. 81. Cell; 1995. p. 289–98. [7] Gonzalez-Perrett S, Kim K, Ibarra C, et al. Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel. Proc Natl Acad Sci U S A 2001;98:1182–7. [8] Koulen P, Cai Y, Geng L, et al. Polycystin-2 is an intracellular calcium release channel. Nat Cell Biol 2002;4:191–7. [9] Torra R, Darnell A, Estivill X, Botey A, Revert L. Interfamilial and intrafamilial variability of clinical expression in ADPKD. Contrib Nephrol 1995;115:97–101. [10] Hateboer N, v Dijk MA, Bogdanova N, et al. Comparison of phenotypes of polycystic kidney disease types 1 and 2. European PKD1-PKD2 Study Group. Lancet 1999;353:103–7. [11] Wu G, Markowitz GS, Li L, et al. Cardiac defects and renal failure in mice with targeted mutations in Pkd2. Nat Genet 2000;24:75–8. [12] Lu W, Peissel B, Babakhanlou H, et al. Perinatal lethality with kidney and pancreas defects in mice with a targetted Pkd1 mutation. Nat Genet 1997;17:179–81. [13] Kim K, Drummond I, Ibraghimov-Beskrovnaya O, Klinger K, Arnaout MA. Polycystin 1 is required for the structural integrity of blood vessels. Proc Natl Acad Sci U S A 2000;97:1731–6. [14] Boulter C, Mulroy S, Webb S, Fleming S, Brindle K, Sandford R. Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene. Proc Natl Acad Sci U S A 2001;98: 12174–9. [15] Pennekamp P, Karcher C, Fischer A, et al. The ion channel polycystin2 is required for left–right axis determination in mice. Curr Biol 2002;12:938–43. [16] Chapman AB, Johnson AM, Rainguet S, Hossack K, Gabow P, Schrier RW. Left ventricular hypertrophy in autosomal dominant polycystic kidney disease. J Am Soc Nephrol 1997;8:1292–7. [17] Kimberling WJ, Fain PR, Kenyon JB, Goldgar D, Sujansky E, Gabow PA. Linkage heterogeneity of autosomal dominant polycystic kidney disease. N Engl J Med 1988;319:913–8. [18] Stypmann J, Engelen MA, Epping C, et al. Age and gender related reference values for transthoracic Doppler-echocardiography in the anesthetized CD1 mouse. Int J Card Imaging 2006. [19] Petermann I, Mayer C, Stypmann J, et al. Lysosomal, cytoskeletal, and metabolic alterations in cardiomyopathy of cathepsin L knockout mice. FASEB J 2006;20:1266–8. [20] Stypmann J, Janssen PM, Prestle J, et al. LAMP-2 deficient mice show depressed cardiac contractile function without significant changes in calcium handling. Basic Res Cardiol 2006;101:281–91. [21] Strauch OF, Stypmann J, Reinheckel T. Cardiac and ocular pathologies in a mouse model of mucopolysaccharidosis type VI. Pediatr Res 2003;54:701–8. [22] Collins KA, Korcarz CE, Shroff SG, et al. Accuracy of echocardiographic estimates of left ventricular mass in mice. Am J Physiol Heart Circ Physiol 2001;280:H1954–62. [23] Ghanem A, Röll W, et al. Echocardiographic assessment of left ventricular mass in neonatal and adult mice — Accuracy of different echocardiographic methods. Echocardiography 2006;23(10):900–7. [24] Tiemann K, Weyer D, Djoufack PC, et al. Increasing myocardial contraction and blood pressure in C57BL/6 mice during early postnatal development. Am J Physiol Heart Circ Physiol 2003;284:H464–74. [25] Fick GM, Johnson AM, Hammond WS, Gabow PA. Causes of death in autosomal dominant polycystic kidney disease. J Am Soc Nephrol 1995;5:2048–56. [26] Perrone RD, Ruthazer R, Terrin NC. Survival after end-stage renal disease in autosomal dominant polycystic kidney disease: contribution of extrarenal complications to mortality. Am J Kidney Dis 2001;38: 777–84. [27] Ecder T, Schrier RW. Hypertension in autosomal-dominant polycystic kidney disease: early occurrence and unique aspects. J Am Soc Nephrol 2001;12:194–200.
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[40] Chung TK, Chen KS, Yen CL, Chen HY, Cherng WJ, Fang KM. Acute abdomen in a haemodialysed patient with polycystic kidney diseaserupture of a massive liver cyst. Nephrol Dial Transplant 1998;13: 1840–2. [41] Adeola T, Adeleye O, Potts JL, Faulkner M, Oso A. Thoracic aortic dissection in a patient with autosomal dominant polycystic kidney disease. J Natl Med Assoc 2001;93:282–7. [42] Belz MM, Hughes RL, Kaehny WD, et al. Familial clustering of ruptured intracranial aneurysms in autosomal dominant polycystic kidney disease. Am J Kidney Dis 2001;38:770–6. [43] Lantinga-van Leeuwen IS, Dauwerse JG, Baelde HJ, et al. Lowering of Pkd1 expression is sufficient to cause polycystic kidney disease. Hum Mol Genet 2004;13:3069–77. [44] Qian Q, Hunter LW, Li M, et al. Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells. Hum Mol Genet 2003;12:1875–80. [45] Peters DJ, Breuning MH. Autosomal dominant polycystic kidney disease: modification of disease progression. Lancet 2001;358: 1439–44. [46] Roth DM, Swaney JS, Dalton ND, Gilpin EA, Ross Jr J. Impact of anesthesia on cardiac function during echocardiography in mice. Am J Physiol Heart Circ Physiol 2002;282:H2134–40. [47] Yang XP, Liu YH, Rhaleb NE, Kurihara N, Kim HE, Carretero OA. Echocardiographic assessment of cardiac function in conscious and anesthetized mice. Am J Physiol 1999;277:H1967–74. [48] Desai KH, Sato R, Schauble E, Barsh GS, Kobilka BK, Bernstein D. Cardiovascular indexes in the mouse at rest and with exercise: new tools to study models of cardiac disease. Am J Physiol 1997;272: H1053–61. [49] Esposito G, Santana LF, Dilly K, et al. Cellular and functional defects in a mouse model of heart failure. Am J Physiol Heart Circ Physiol 2000;279:H3101–12. [50] Semeniuk LM, Kryski AJ, Severson DL. Echocardiographic assessment of cardiac function in diabetic db/db and transgenic db/dbhGLUT4 mice. Am J Physiol Heart Circ Physiol 2002;283:H976–82.
Cardiovascular characterization of Pkd2 +/LacZ mice, an animal model for the autosomal dominant polycystic kidney disease type 2 (ADPKD2) Jörg Stypmann a,b,1 , Markus A. Engelen a,c,1 , Stefan Orwat a , Konstantinos Bilbilis d , Markus Rothenburger e , Lars Eckardt a , Wilhelm Haverkamp a,b , Jürgen Horst d , Bernd Dworniczak d , Petra Pennekamp d,⁎ b
a Department of Cardiology and Angiology, Hospital of the University of Münster, Germany Interdisciplinary Center for Clinical Research (IZKF), Central Project Group 4 (ZPG4a), Münster, Germany c Department of Medical Physiology, University Medical Center Utrecht, The Netherlands d Institut für Humangenetik, Universitätsklinikum Münster, Vesaliusweg 12, D - 48149 Münster, Germany e Department for Thoracic and Cardiovascular Surgery, Hospital of the University of Münster, Germany
Received 21 February 2006; received in revised form 27 August 2006; accepted 20 September 2006 Available online 19 December 2006
Abstract Background: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2. Patients with ADPKD have an increased incidence of cardiac valve abnormalities and left ventricular hypertrophy. Systematic analyses of cardiovascular involvement have so far been performed only on genetically unclassified patients or on ADPKD1 patients, but not on genetically defined ADPKD2 patients. Even existing Pkd1 or Pkd2 mouse models were not thoroughly analyzed in this respect. Therefore, the aim of this project was the noninvasive functional cardiovascular characterization of a mouse model for ADPKD2. Methods: Pkd2+/LacZ mice and wildtype controls were classified into 8 groups with respect to gender, age and genotype. In addition, two subgroups of female mice were analyzed for cardiac function before and during advanced pregnancy. Doppler-echocardiographic as well as histological studies were performed. Results: Doppler-echocardiography did not reveal significant cardiovascular changes. Heart rate and left ventricular (LV) length, LV mass, LV enddiastolic and LV endsystolic diameters did not differ significantly among the various groups when comparing wildtype and knockout mice. There were no significant differences except for a tendency towards higher maximal early and late flow velocities over the mitral valve in old wildtype mice. Conclusions: Non-invasive phenotyping using ultrasound did not reveal significant cardiovascular difference between adult Pkd2+/LacZ and WT mice. Due to the lack of an obvious renal phenotype in heterozygous mice, it is likely that in conventional ADPKD knock out mouse models severe cardiac problems appear too late to be identified during the reduced lifespan of the animals. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Polycystin-2; Pkd2; APKD; Cystic kidney disease; Ultrasound; Echocardiography
1. Introduction Autosomal dominant polycystic kidney disease (ADPKD) is a frequent hereditary renal disorder with an overall incidence of 1 to 2.5 in 1000 [1]. It is characterized by the ⁎ Corresponding author. Tel.: +49 251 8355405; fax: +49 251 8356995. E-mail address: [email protected] (P. Pennekamp). 1 The first two authors contributed equally to this work. 0167-5273/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2006.09.013
formation of multiple, fluid-filled cysts not only in both kidneys but also in other organs [2]. About 50% of PKD patients develop end-stage renal failure (ESRF) by late middle age [3], accounting for 5–10% of patients requiring haemodialysis world wide [4]. However, the course of the disease frequently is complicated by a multiplicity of extra renal manifestations: even in patients with normal renal function, nearly 70% suffer from hypertension; many patients develop left ventricular hypertrophy and 20% suffer from
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intracranial aneurysm. Furthermore, patients can be affected by cardiac valvular abnormalities, colonic diverticuli, and inguinal hernia. In the meantime, ADPKD is regarded as a multisystemic disorder [5]. So far, two genes have been identified which are mutated in the majority (99%) of ADPKD cases: PKD1, encoding polycystin-1 (PC1), an integral membrane-spanning glycoprotein suggested to function as epithelial cell membrane mechanoreceptor, sensing morphogenetic cues in the extracellular environment, and PKD2, encoding polycystin-2 (PC2), a membrane-spanning protein as well functioning as a Ca2+ permeable cation channel [6–8]. Both polycystins are likely to function in a common pathway because mutations in either gene cause strikingly similar phenotypes. Although the disease displays inter- and intrafamilial phenotypic heterogeneity [9], detailed studies revealed that in the long run ADPKD has a milder course in families linked to PKD2. These patients suffer later from symptoms, exhibit a smaller number of cysts at the time of diagnosis, and chronic renal failure threaten PKD2 patients later in life as compared to PKD1 patients [10]. Orthologues of the human PKD1 and PKD2 genes exist in the mouse genome, and several different knockout mice have been created. Heterozygous mice appear normal but develop single cysts in the kidney or liver late in life and have a reduced overall lifespan [11]. Homozygous null mutant mice die in utero with massively enlarged cystic kidneys, pancreatic ductal cysts and pulmonary hypoplasia and often exhibit edema, vascular leaks, and rupture of blood vessels suggesting that the polycystins are required for the structural integrity of blood vessels [12,13]. In addition most of the homozygous embryos display multiple cardiac abnormalities including cardiac septation defects, double outlet right ventricle and pericardial effusions [11,14,15]. Severe cardiovascular abnormalities in homozygous mutant PKD1 or PKD2 knockout mouse embryos might be due to loss of polycystin function in the heart tissue which normally shows a high expression of both genes (Figs. 2 and 3; [14]). Moreover, patients frequently develop left ventricular hypertrophy before developing hypertension [16]. Therefore, the cardiovascular complications in ADPKD patients begun to be recognized as a primary defect due to the loss of PC1 and/or PC2 function in cardiovascular organs and not solely as a secondary defect due to decreasing renal function. However, owing to the similarity of the overall clinical phenotypes of ADPKD1 and 2 patients, it is assumed that the cardiovascular phenotypes in ADPKD1 and ADPKD 2 patients are virtually identical [17]. But, systematic analyses of the cardiovascular involvement have so far been performed only on genetically unclassified patients or on ADPKD1 patients, but not on genetically well defined panels of ADPKD2 patients. Even existing Pkd1 or Pkd2 mouse models were not thoroughly analyzed in that respect. Therefore, in the present study the cardiovascular performance of the Pkd2+/LacZ mouse as a mouse model for the ADPKD2 was investigated in more detail using noninvasive Doppler-
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echocardiography of heterozygous and wildtype mice of different gender and age. In addition, two subgroups of mice before and during advanced pregnancy were examined to clarify whether pregnancy deteriorates the heart performance in this mouse model of ADPKD2. 2. Methods 2.1. Generation and maintenance of Pkd2-deficient mice Pkd2-deficient mice have been generated by gene targeting in mouse embryonic stem cells as described previously [15]. In this mouse line, we replaced Pkd2 exon 1 and part of intron 1 by LacZ as a reporter gene. LacZ was fused to the original Pkd2 ATG start codon enabling to monitor LacZ expression under the control of the Pkd2 promotor (Mouse Genome Informatics: allele Pkd2tm1Dwo (http://www.informatics.jax. org). In this report, heterozygous Pkd2 mutated mice are named Pkd2+/LacZ and homozygous Pkd2 mutated mice are named Pkd2LacZ/LacZ). For the present study, Pkd2+/LacZ mice on a mixed C57/BL6 and 129/Sv genetic background were used. Mice were conventionally housed in a controlled environment at a temperature of 21 °C ± 1 and a 12 h day/night cycle. Mice were kept in macrolon cages and had access to standard food pellets and tap water ad libidum. All experimental procedures had been approved by the local government authorities and confirm to the NIH Guide for the Care and Use of Laboratory Animals. 2.2. Expression analysis by LacZ staining Whole embryos were fixed in 4% PFA/PBS + 2 mM MgCl2 for 5–30 min at room temperature and washed 3 times in 1 × PBS, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40. LacZ staining was performed by incubation of samples with 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 3H2O, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, and 1 mg/ml X-Gal (4-chloro-5-bromo-3-indolyl-β-Dgalactopyranoside) in 1 × PBS (pH 7.4–8.0) at 30 °C. After staining, samples were washed in 1 × PBS, postfixed in 4% PFA, dehydrated in methanol, and embedded in Historesin (Leica Microsystems, Bensheim, Germany). Stained embryos were sectioned at 4 μm on a MICROM HM 355 microtome (MICROM, Walldorf, Germany) and staining was visualized under darkfield illumination by use of a Zeiss Axioskop microscope. Adjacent sections were counterstained with methylen blue/azur II and visualized under bright field illumination. Adult organs were frozen and sectioned on a MICROM HM 500 O microtome (MICROM, Walldorf, Germany) at 20 μm for X-Gal staining as described above and counterstained with eosin. 2.3. Grouping of mice for analysis Pkd2+/LacZ mice and their wildtype littermates were classified into 10 groups with respect to gender, age and genotype as
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Table 1 Basic characteristics of all Doppler-echocardiographically examined mice Group
I
Age
102 ± 33 days
II
Gender
Female
Genotype Number of animals Body weight [g] Heart rate [bpm]
Pkd2+/LacZ 21 22.1 335 ± 58
Ib
IIb
III
IV
V
VI
VII
VIII
483 ± 69 days
WT 23 22.3 322 ± 70
Female/pregnant
Male
Pkd2+/LacZ 7 33.79 387 ± 145
Pkd2+/LacZ 6 28.6 333 ± 74
WT 5 33.04 356 ± 38
shown in Tables 1 and 2. In addition, after the first examination several female mice out of groups I and II were mated to analyze cardiac performance during pregnancy between embryonic day (E) 15 and E17 (subgroups Ib and IIb). The age of the young animals (groups I–IV) varied between 2 and 5 months (mean age 102 ± 33 days), the age of the old animals (groups V–VIII) varied between 14 and 16 months (mean age 483 ± 69 days). 2.4. Doppler-echocardiography Doppler-echocardiographic examination was performed as previously described in detail [18–20]. The investigator
Female WT 11 30.0 353 ± 70
Male
Pkd2+/LacZ 6 38.3 346 ± 33
WT 6 38.6 312 ± 95
Pkd2+/LacZ 6 40.9 357 ± 48
WT 6 41.0 323 ± 46
was blinded with regard to the genotype of the mice. After induction of light anesthesia with a combination of 50 mg/kg ketamin (Ketavet®, Pfizer Pharma GmbH, Karlsruhe, Germany) and 5 mg/kg xylazine (Rompun®, BayerVital, Leverkusen, Germany), the chests of the mice were precordially shaved. The mice were secured in a supine position to a warm waterbed heated to 38 °C and angled 45° to the left. One lead ECG was used to monitor heart rate. Transthoracic echocardiography was performed using a commercially available digital cardiac ultrasound platform equipped with either a 6- to 12-MHz short focal length-phased array transducer equipped with an acoustic stand-off or a 15-MHz linear-array transducer (Sonos 5500, B1-software package,
Table 2 Doppler-echocardigraphic data of left ventricular performance with subgroup analyses Group
I
Age
102 ± 33 days
II
Gender
Female +/LacZ
Female/pregnant
Pkd2 1.99 ± 0.31 0.82 ± 0.07 4.29 ± 0.91 2.76 ± 0.71 0.88 ± 0.09 5.97 ± 0.45 35.08± 6.80
WT 1.96 ± 0.38 0.85 ± 0.11 4.28 ± 1.13 2.68 ± 0.68 0.94 ± 0.10 6.00 ± 0.76 37.04 ± 5.96
LV-EF [%] Mass of LV [mg] LV mass index [mg/g] LV-CO [ml/min] Cardiac index [ml/min/g] Ao Vmax [cm/s] Ao PGmax [mmHg] Ao MPG [mmHg]
57 ± 9 92 ± 25 4.28 ± 1.32 10 ± 2 0.47 ± 0.13 59.7 ± 6.6 1.46 ± 0.33 0.779 ± 0.184 42.7 ± 8.8 21.3 ± 5.2
60 ± 8 100 ± 33 4.50 ± 1.63 11 ± 3 0.50 ± 0.16 60.8 ± 6.0 1.49 ± 0.30 0.793 ± 0.176 47.2 ± 10.3 23.6 ± 8.0
2.08 ± 0.53 0.271 ± 0.093
2.12 ± 0.57 0.300 ± 0.146
Ratio of E/A-Wave MV PGmean [mmHg]
IIb
III
IV
V
VI
VII
VIII
483 ± 69 days
Genotype LA [mm] LVAWd [mm] LVEDd [mm] LVESd [mm] LVPWd [mm] LV-length [mm] LV-FS [%]
MV E-wave [cm/s] MV A-wave [cm/s]
Ib
+/LacZ
Pkd2 2.14 ± 0.33 0.88 ± 0.04 4.00 ± 0.83 2.35 ± 0.65 0.66 ± 0.05 6.67 ± 0.66 42.05± 4.56 66 ± 6 100 ± 19 2.98 ± 0.51 18 ± 4 0.53 ± 0.13 68.30 ± 7.2 1.89 ± 0.40 0.96 ± 0.750 48.14 ± 6.8 30.46 ± 16.6 1.93 ± 0.68 0.33 ± 0.080
WT 2.02 ± 0.37 0.82 ± 0.06 3.75 ± 0.63 2.20 ± 0.31 0.64 ± 0.07 6.53 ± 0.48 40.67 ± 5.97 64 ± 7 91 ± 9 2.77 ± 0.31 17 ± 1 0.53 ± 0.03 68.94 ± 7.2 1.93 ± 0.40 0.75 ± 0.340 55.94 ± 6.5 29.04 ± 7.76 2.01 ± 0.33 0.40 ± 0.140
Male +/LacZ
Pkd2 1.90 ± 0.14 0.77 ± 0.08 3.43 ± 0.29 1.89 ± 0.31 0.84 ± 0.10 6.46 ± 0.79 45.04 ± 6.75 69 ± 8 74 ± 13 2.60 ± 0.34 14 ± 3 0.49 ± 0.12 77.1 ± 13.5 2.44 ± 0.82 1.239 ± 0.390 58.7 ± 11.8 25.8 ± 5.5
Female WT 2.02 ± 0.30 0.79 ± 0.09 4.00 ± 0.85 2.36 ± 0.63 0.86 ± 0.11 6.57 ± 0.28 41.50 ± 6.79 65 ± 8 90 ± 19 2.99 ± 0.55 15 ± 4 0.50 ± 0.14 75.0 ± 8.9 2.28 ± 0.57 1.230 ± 0.315 62.8 ± 14.1 30.7 ± 8.9
+/LacZ
Pkd2 1.84 ± 0.29 0.91 ± 0.10 2.85 ± 0.25b 1.92 ± 0.18b 1.05 ± 0.14b 6.01 ± 0.45 32.39 ± 5.88 54 ± 8 78 ± 17 1.97 ± 0.38b 17 ± 6b 0.42 ± 0.10 58.6 ± 5.8 1.39 ± 0.28 0.736 ± 0.180 41.1 ± 4.4 20.6 ± 3.5
2.33 ± 0.51 2.09 ± 0.35 2.05 ± 0.47 0.372 ± 0.472 ± 0.295 ± 0.078 0.147 0.123
Male WT 1.80 ± 0.09 0.94 ± 0.11 3.05 ± 0.14 2.08 ± 0.04 1.02 ± 0.08a 5.90 ± 0.63 31.72 ± 2.03 53 ± 3 81 ± 14 2.12 ± 0.39c 14 ± 3 0.35 ± 0.08 62.0 ± 3.8 1.54 ± 0.19 0.837 ± 0.097 39.7 ± 7.6 20.8 ± 4.9
Pkd2+/LacZ 2.00 ± 0.39 0.94 ± 0.06d 3.10 ± 0.26 1.90 ± 0.15 1.02 ± 0.07 6.27 ± 0.61 38.38 ± 4.57 62 ± 6 86 ± 8 2.14 ± 0.37 16 ± 2 0.41 ± 0.13 63.6 ± 1.6 1.62 ± 0.09 0.841 ± 0.076 54.5 ± 13.3 18.1 ± 6.3
WT 2.12 ± 0.22 1.01 ± 0.13e 3.26 ± 0.34 1.84 ± 0.21 0.97 ± 0.11 6.09 ± 0.61 43.38 ± 4.39 68 ± 5 91 ± 22 2.42 ± 0.78 16 ± 3 0.43 ± 0.14 75.8 ± 9.1 2.33 ± 0.53 1.123 ± 0.285 57.3 ± 4.5 24.0 ± 3.8
1.95 ± 0.28 0.215 ± 0.070
3.21 ± 1.09 0.375 ± 0.214
2.43 ± 0.35 0.364 ± 0.088
Ao PGmax: aortic maximum pressure gradient [mmHg]; Ao MPG: mean aortic pressure gradient [mmHg]; Ao Vmax: aorta velocity maximum [cm/s]; LVAWD: LV anterior wall enddiastolic diameter; LVPWd: LV posterior wall enddiastolic diameter; LA: left atrial diameter; LV: left ventricle; LVEDD: left ventricular enddiastolic diameter; LV-EF: LV-ejection fraction; LVESD: left ventricular endsystolic diameter; LV-FS: LV-fractional shortening; LVOT-CO: Dopplerechocardiographic cardiac output via left ventricular outflow tract; MV A-Wave: atrial inflow of mitral valve; MV E-Wave: early inflow of mitral valve; MV PGmean: Mean pressure gradient of mitral valve. Significances p b 0.05: a = V/VI, b = I/V, c = II/VI, d = III/VII, e = IV/VIII.
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Fig. 1. Representative echocardiographic images. Parasternal long axis view (A), M-mode (B), Doppler flow signals of the mitral inflow with E- and A-wave (C) and Doppler flow over the aortic valve (D). Velocity of the Doppler flow signals shown on the right side of the images are given in m/s. Exemplary image of an aortic insufficiency (E). LV = left ventricle, LA = left atrium, Ao = aorta.
Philips, The Netherlands). A smooth layer of 1.5–2 cm thick centrifuged ultrasound gel was placed on the chest of the mice and the probe was gently dipped into this layer of gel avoiding any pressure on the thorax to prevent thorax deformation or reflex bradycardia. Parasternal long-axis and short-axis views were obtained at a frame rate of 300 per second. The variable depth of the zoom box was set at 2– 4 cm. M-mode and Doppler recordings were performed at a sweep speed of 150 mm/s. M-Mode data of the LV anterior (LVAWd) and posterior wall thickness (LVPWd) at enddiastole as well as end-diastolic and end-systolic dimensions of the left ventricle were acquired in the parasternal long axis view following the leading edge to leading edge method [20] of three consecutive cycles. Percentage of fractional shortening (FS) as an index of LV systolic function was calculated using the equation [18,20–22] FSð%Þ ¼
LVEDD−LVESD d100: LVEDD
Echocardiographic LV mass was calculated using the arealength method as previously described [22–24] and corrected for bodyweight by calculating the LV mass index [18]. Pulsed wave Doppler signals were obtained in a modified apical view by placing the sample volume parallel to the flow into LV outflow tract (LVOT) for systolic outflow of the left ventricle as well as apical to the mitral valve within left ventricle for diastolic inflow. The left atrium was checked for mitral regurgitation by pulsed wave Doppler. In addition, mitral and aortic valves were examined for regurgitation by color flow Doppler. Cardiac output was calculated using
heart rate, velocity time integral of the Doppler-signals in the LV outflow tract and the end-diastolic diameter of the LVOT [21]. 3. Histological analyses For light microscopy, organs were fixed in 4% paraformaldehyde dissolved in 1 × PBS (4% PFA/PBS) solution and embedded in paraffin. Sections of 6 μm were stained with methylen blue/azur II and analyzed by light microscopy (Axioskop, Zeiss, Germany). 4. Statistical analyses Values were calculated with SPSS 11 statistical software and expressed as means and their respective standard errors or standard deviations. Groups I/II, I/Ib, II/IIb, Ib/IIb, III/IV, V/VI, VII/VIII, I/V, II/VI, III/VII and IV/VIII were compared using the two-tailed Student's t test and the Wilcoxon rank sum test. A p b 0.05 was considered to indicate statistically significant differences between the groups investigated. 5. Results 85 mice were included in this study including 12 female mice which were examined before and during pregnancy. As expected, the body weight of female non-pregnant mice (25.9 ± 8.4 g) was significant lower than the body weight of male mice (33.8 ± 6.8 g) (p b 0.0001) and body weight increased with age in both genders. No significant differences of body weight or heart rate have been observed between
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Fig. 2. Pkd2 expression in the heart of adult Pkd2+/LacZ mice. Pkd2 expression is reduced in the cardiac region of adult mice, but still can be detected in the cardiac valves and the septum membranaceum as demonstrated by X-Gal histochemistry (blue dots in A I, A II, B I and B II). Frontal sections of a heart isolated from a Pkd2+/LacZ mouse (A and B) and higher magnifications of the aortic valve (A I), tricuspid valve (A II), mitral valve (B I) and septum membranaceum (B II) viewed under bright field illumination.
Fig. 3. Pkd2 expression and abnormal cardiovascular development in Pkd2LacZ/LacZ embryos and a representative kidney section of a male Pkd2+/LacZ mouse at the age of 16 month. In this mouse line, LacZ is fused to the Pkd2 ATG start codon enabling to monitor LacZ expression under the control of the Pkd2 promotor using whole mount X-Gal staining. Viewed under darkfield illumination, blue X-Gal precipitates appear pink. Expression to different extend was detectable in all structures of the heart with the highest expression in the outflow tracts, especially the endocardial cushions and valve leaflets, and lower levels in the myocardium. Mutant embryos showed cardiovascular abnormalities to different extend including disorganization of the myocardial wall and ventricular septum, dysplastic endocardial cushions, double outlet right ventricle (DORV) and single chambered ventricle. Frontal (A–F) and sagital sections of WT (A), Pkd2+/LacZ (B, G) and Pkd2LacZ/LacZ (C–F, H) embryonic hearts and kidney section of a male Pkd2+/LacZ mouse at the age of 16 month (I). A, B: newborn; C, E– H: E16.5; D: E17.5; I: E15.5; A–F: X-Gal staining viewed under dark field illumination; G–I: bright field illumination; G, H, I: methylen blue/azur II staining.
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Pkd2+/LacZ and wildtype mice of the same age and gender (Table 1). Heart rate did not differ significantly between all groups. The anesthetic regimen did not produce significant, prolonged or temporally variable changes in heart rate during the examination. Adequate Doppler-echocardigraphic measurements could be obtained in all mice without any problems within less than 20 min (mean less than 15 min). Representative images are shown in Fig. 1. Dopplerechocardiographic measurements and calculated values are shown in Table 2. Overall, there were not much striking significant differences between the compared groups. Color flow Doppler revealed insufficiencies of the aortic valve in one young female Pkd2+/LacZ mouse (group I) and in one young male wildtype mouse (group IV). LVEDD and LVEDS were significantly decreased in the old female Pkd2+/LacZ mice as compared with young heterozygous knockout mice. LVPWd and CO increased significantly with age in the female Pkd2+/LacZ mice. LVAWd was significantly increased as well in the old male Pkd2+/LacZ mice as in the old wildtypes as compared with younger mice of the respective genotype and gender. Ao Vmax decreased significantly with age in the Pkd2+/LacZ mice. FS, Ewave and A-wave were higher in male mice independent of the genotype when compared to females of the same age. Both gender and genotypes showed a tendency to an increase in cardiac output at older ages, but this trend cleared out after correction for body-weight. The observed time pattern of sonomorphometric indices during pregnancy and changes in functional Doppler-echocardiographic measurements in the examined mice did not show significant differences between Pkd2+/LacZ and wildtype mice. In summary, there were no significant differences between age-matched Pkd2 heterozygous mice and wildtype controls. As previously shown, embryos lacking polycystin-2 (Pkd2LacZ/LacZ) exhibited severe defects in cardiac morphogenesis including ventricular septal defects (VSD), abnormal formation of cardiac valves and transposition of the great vessels (TGA) [15,11]. During mouse embryogenesis, polycystin-2 is, similar to polycystin-1 [14], highly expressed in the conotruncus, especially in the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. In adult animals, expression of polycystin2 is decreased in the cardiac region but still can be detected in the cardiac valves and the septum membranaceum (Fig. 2). In the kidney, only few cysts can be detected in adult heterozygous knockout mice older than one year (Fig. 3). As expected, renal function in these mice is not affected ([11] and unpublished own data). 6. Discussion In recent years, autosomal dominant polycystic kidney disease (ADPKD) became increasingly recognized as a systemic disorder and on the cellular level this seems to
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be reasonable due to the wide spread expression of both genes. Although ADPKD patients suffer from many extrarenal manifestations [2], cardiovascular problems are the most severe side effects and several studies have been conducted to characterize the cardiac involvement in ADPKD patients. Cardiovascular problems seem to be a continuous process starting early during the course of the disease and it has to be pointed out that they are still a major cause of morbidity and mortality in patients with ADPKD [25,26]. Hypertension and left ventricular hypertrophy is common and associated with faster progression to end stage renal disease [16,25,27]. Especially hypertension develops in the majority of patients before any decrease in renal function can be determined [27]. It has been proposed that formation and expansion of renal cysts activate the renin–angiotensin– aldosterone system (RAAS) by which development of hypertension is triggered [28]; consistent with this hypothesis activation of RAAS can be verified well before onset of hypertension or any other clinical finding in ADPKD patients [29,30]. Recent studies showed that patients benefit from lowering the mean arterial pressure and by use of angiotensin-converting enzyme inhibitors which significantly slow down the decrease of renal function [31]. Beside hypertension and left ventricular hypertrophy, the prevalence of mitral-valve prolapse, mitral regurgitation, aortic insufficiency, tricuspid incompetence, and tricuspidvalve prolapse is increased [32]. The same holds true for the left ventricular mass (LVM) index and it was clearly shown that left ventricular dysfunction increases if ADPKD patients become hypertensive [33]. The same study revealed that the majority of valvular abnormalities occurred in dialysis patients which were generally related to annular calcification of the mitral or aortic valve. However, despite the feasibility of subdividing ADPKD patients into genetically defined groups (ADPKD type I caused by mutations in PKD1 versus ADPKD type 2 caused by mutation in PKD2) up to now only a single study has been published comparing a genetically classified cohort of ADPKD type 1 patients to unaffected family members and healthy controls [34]. In addition, although Pkd1 and Pkd2 mouse models are available, there are to the best of our knowledge no published data concerning the question whether cardiac performance is impacted in these animals. But, embryos lacking polycystin-2 and some of the mouse mutants for polycystin-1 [11,14,15] exhibit severe defects in cardiac morphogenesis including ventricular septal defects (VSD), abnormal formation of cardiac valves and transposition of the great vessels (TGA). Furthermore, during mouse embryogenesis, polycystin-2 is highly expressed in the conotruncus, especially the endocardial cushions, structures that give rise to the affected heart valves and the membranous ventricular septum. Later in development, expression of the polycystins can still be detected in the cardiac valves and great vessels. The prominent cardiac expression patterns for both polycystins suggest a relevant function of
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both proteins not only during development but in addition for maintenance of cardiac valves during adulthood. These findings let us assume that Pkd knockout mice can serve as model to analyze cardiac involvement in ADPKD in more detail. As initial step we therefore performed a noninvasive analysis of the cardiac performance of heterozygous mice of different gender and age using Doppler-echocardiography. In addition, two subgroups of female mice were analyzed for cardiac function before and during advanced pregnancy. Our echocardiographic data correspond very well with previously published values obtained in similar analyses using C57/Bl6 mice [24,35]. Despite the assumed function of the polycystins during development of cardiac structure and during adulthood, we did not detect significant pathological cardiovascular changes neither in young nor in older Pkd2+/LacZ mice of either gender or during pregnancy. Particularly, there were hardly any differences in respect to wall thickness, LV mass and occurrence of valvular insufficiencies. The significant change in LV mass index of the young versus the old female mice is due to changes of the body-weight during aging. If Pkd2+/LacZ mice had developed arterial hypertension, we would expect significant differences in left ventricular mass due to different load, which was not the case. These surprising findings might be due to a couple of different reasons: The course of the disease of ADPKD1 or ADPKD2 patients is in general clinically hardly to distinguish, but long term investigations uncovered that disease progression is milder in ADPKD2. Exemplary, onset of end-stage renal disease is later and ADPKD2-patients are less likely to have hypertension, urinary-tract infection or hematuria [10] and the same might be true for cardiovascular problems. However, following the course of the disease in human patients, independent whether it is caused by PKD1 or PKD2 mutations, severity of most symptoms clearly progress with increasing age of affected patients. Assumed that progression of disease is comparable fast in humans and mice, this ageing effect is only mimicked partially in mice. Cyst formation in kidney should serve as an example: Even though the mutational mechanism underlying initiation of cyst formation in ADPKD remains controversial the most widely accepted hypothesis proposes a two-hit mechanism of cyst formation to explain the focal nature of cyst development. However, frequency of the second hit clearly depends on time and total number of renal cells susceptible for the loss [36–38]. Indeed, cyst formation in conventionally targeted Pkd1 and Pkd2 mice is only detected rarely and their number and relative size is reduced as compared to humans [11] and resulting reduction of the renal function in these mice is obviously too low to impact cardiac function. Nevertheless, approaching the age of 15–18 months, many Pkd2+/LacZ mice suddenly die ([11] and unpublished own observations). Macroscopic investigations of deceased animals – if possible – suggest that death was either caused by ruptures of blood vessels or of large liver cysts similar to what is described for humans [39,40,25,41,42].
However, we could not identify the reason for sudden death in all animals investigated. Nevertheless, data form us and other groups show that haploinsufficiency of either Pkd1 or Pkd2 is sufficient to reduce survival of mutant mice [11,43,44]. Interestingly, it has been shown that loss of Polycystin 1 or 2 function and haploinsufficiency of either Pkd1 or Pkd2 deteriorates the structural integrity of the blood vessel walls [13,43,44] suggesting that sudden ruptures of these vessels might be a major cause of sudden death in these animals. But, if decreased survival of mice for which we could not identify the cause of death would be caused by cardiac failure, cardiovascular abnormalities have to develop very rapid because in part of this study we examined a rather old mouse population (14–16 months old) short before animals reached the end of the average life span and did not find gross structural or functional changes explaining forthcoming sudden death. If cardiac involvement in ADPKD is not only secondary but due to loss of polycystin function in the heart tissue itself – as we hypothesize – again average life span of Pkd2+/LacZ mice seems to be too short to emerge. Thus, these changes are hardly to detect in commonly sized cohorts of mice. Even triggering the development of cardiovascular problems by additional stress – in our case we measured heart performance before and during pregnancy – did not uncover pathological findings. It is known that in human disease progression is influenced by the type of the germ line and somatic mutations and so far unknown modifier genes. Especially in animal models it could be shown that environmental factors e.g. diet and housing conditions, could also affect disease progression [45]. To exclude an influence of the environmental factors on disease progression, especially housing and diet were kept constant during the complete study. Data from the literature and own observations suggest that in mice modifier genes play an important role in disease progression. In case of Pkd2 this indeed results in a more severe phenotype in homozygous mutant embryos on a 129/Sv genetic background compared to a pure C57/BL6 genetic background (unpublished own observation) leading to an earlier prenatal death in 129/Sv embryos. In this study, we examined F1 and N2 mice derived from backcrosses between 129/Sv (donor strain) and C57/BL6 (recipient strain) with the F1 generation providing especially the mice of the 14–16 month old mouse population. Though we cannot exclude the influence of modifier genes on the cardiac performance of heterozygous Pkd2 mutant mice, at least in the genetically homogenous F1 generation, the influence of these modifier genes should have been minimized if not excluded. During the complete study environmental factors, especially housing and diet, were kept constant to eliminate any possible influence of these factors. This tightly controlled study enabled us to evaluate solely the influence of haploinsufficiency of the Pkd2 mutation on to cardiac performance and resulting data can be used as base line for future studies.
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7. Limitations One limitation of this study is the low medium heart rate due to the use of ketamin–xylazine–narcosis. Most available narcotics have an impact on cardiac function of the mouse [46,47], partly by their influence on autonomic tone, partly due to a direct cardiodepressive effect. The normal heart rate of mice lies around 475–540 bpm [48] but heart rate is clearly depressed by most anesthetic drugs. Anesthesia by inhalation of volatile anesthetics like isoflurane or sevoflurane has less impact on cardiac function and heart rate. Some groups have performed echo in awake mice [47,49,50], but this technique requires a lot of training of the animals, a second researcher and much more time. Furthermore, this technique causes a high level of stress for the mice which again changes cardiac function and especially will increase heart rate. Finally, a change of cardiac morphology by fixing the mouse in a supine position cannot be excluded. 8. Conclusion In conclusion, the cardiac performance of our mouse model for Pkd2 seems to be not affected by partial loss of polycystin 2 function. This diagnostic finding is likely due to the fact that the life span of mice is too short to develop cardiovascular problems. Although it could be of some interest to analyze the cardiovascular phenotype of the different Pkd1 knockout mouse models we do not expect tremendous differences compared to our result. Acknowledgement Parts of these data are part of the doctoral thesis of Stefan Orwat. We thank Axel Bohring for helpful discussions. This work was partly supported by the Interdisciplinary Center for Clinical Research (IZKF) Münster, project numbers ZPG4a and BD IKF 2B 4 and by grants from the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich 656 MoBil Münster, Germany (project C3). Recent address of Wilhem Haverkamp: Medizinische Klinik mit Schwerpunkt Kardiologie, Campus VirchowKlinikum Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D – 13353 Berlin, Germany. References [1] Gabow PA. Autosomal dominant polycystic kidney disease. N Engl J Med 1993;329:332–42. [2] Gabow PA. Autosomal dominant polycystic kidney disease—more than a renal disease. Am J Kidney Dis 1990;16:403–13. [3] Gabow PA, Johnson AM, Kaehny WD, et al. Factors affecting the progression of renal disease in autosomal-dominant polycystic kidney disease. Kidney Int 1992;41:1311–9. [4] Wilson PD. Polycystic kidney disease. N Engl J Med 2004;350: 151–64. [5] Perrone RD. Extrarenal manifestations of ADPKD. Kidney Int 1997;51:2022–36.
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