Prenatal evaluation of kidney function in mice using dynamic contrast-enhanced magnetic resonance imaging

(1)

O R I G I N A L A R T I C L E

C. ChaponÆ F. FranconiÆJ. Roux J. J. Le JeuneÆ L. Lemaire

Prenatal evaluation of kidney function in mice using dynamic contrast-enhanced magnetic resonance imaging

Accepted: 6 December 2004 / Published online: 15 March 2005 Springer-Verlag 2005

Abstract Glomerular differentiation starts as soon as embryonic stage 12 in mice and suggests that kidneys may be functional at this stage. Dynamic contrast-enhanced magnetic resonance microscopy, a noninvasive imaging technique, was used to assess renal function establishment in utero. Indeed, in adults (n=3), an intravenous injection of gadolinium-DOTA induced in a first step a massive and rapid drop in kidney signal intensity followed, in a second step, by a drop in bladder signal intensity. The delay in signal changes between kidney and bladder reflected glomerular filtration.

Pregnant mice underwent anatomical and dynamic contrast-enhanced magnetic resonance microscopy on postcoital days 12–13 (n=2), 13–14 (n=1), 14–15 (n=3), 15–16 (n=2), 16–17 (n=3), 17–18 (n=3), and 18–19 (n=1). Kidneys and bladder were unambiguously depicted prior to contrast agent injection on stage 15–16 embryos. Contrast agent injection allowed kidney, detection as early as stage 12–13 but not bladder.

Kinetics of signal changes demonstrated that glomerular ﬁltration is established at embryonic stage 15–16 in mice.

Thus, anatomical and dynamic contrast-enhanced magnetic resonance microscopy may be a powerful noninvasive method for in vivo prenatal developmental and functional studies.

Keywords In utero functional imagingÆMRIÆ Contrast agentÆGadolinium Æ EmbryoÆKidney

Introduction

In the fast-developing era of transgenic mouse models, any tool providing information on the eﬀect of gene manipulation is of great interest to the biologist. The mouse (genetically engineered or wild) is the major model system for studying the genetic basis of mam- malian development. Systematic reference description of the phenotype of the mouse embryo is still under establishment using invasive techniques such as histology (Kaufman 1992; Brune et al. 1999) and magnetic resonance (MR) microscopy (Dhenain et al. 2001;

Schneider et al. 2003). In parallel, noninvasive approaches for studying in utero anatomy have been developed to follow up embryos’ development using either ultrasound biomicroscopy (Turnbull et al.1995) or MR microscopy (Smith et al.1998; Chapon et al.2002;

Weintraub et al. 2004). Complementary to these noninvasive anatomical descriptions of mouse embryo gen- esis, functional imaging of the set-up of the biological processes has been used, mainly in addressing ﬂuxes in the embryonic cardiovascular system (Aristiza´bal et al.

1998; Linask and Huhta 2000; Hogers et al.2000).

Addressing renal function is equally of major interest because malformations of the urological tracts account for about 9% of embryonic malformations in humans (Congenital Malformations Australia 1981–97), among which almost 70% correspond to cystic kidney disease, obstructive defects of the renal pelvis/ureter, or stenosis of the urethra.

Theoretically, kidney function could also be assessed in utero using techniques currently available in the clinic, such as ultrasound Doppler, scintigraphy, or dynamic contrast-enhanced magnetic resonance imaging (MRI) (Grattan-Smith et al. 2003). Studies of kidney function in embryonic mice are challenging and require high-resolution imaging because the length of the kidney is about 2 mm by the end of the gestational period.

Contrast agents are required to depict the structure of interest, and rapid imaging is needed in order to evaluate

C. ChaponÆJ. J. Le JeuneÆL. Lemaire (&) INSERM U646 ‘‘Inge´nierie de la Vectorisation’’,

Universite´ d’Angers, 10 rue Boquel, 49100 Angers, France E-mail: laurent.lemaire@univ-angers.fr

Tel.: +33-0241-735006 Fax: +33-0241-735007 F. Franconi

Service Commun d’Analyses Spectroscopiques, Universite´ d’Angers, France

J. Roux

Service Commun Animalerie Hospitalo-Universitaire, Universite´ d’Angers, France

DOI 10.1007/s00429-004-0451-9

(2)

signal evolution post-contrast agent. We have previously set up the technical requirements for such a study in which particular contrast agents were used to attempt to evaluate glomerular ﬁltration, assuming that the agent would penetrate the nonmature kidney but not the mature kidney (Chapon et al. 2002). Unfortunately, the particles tested appeared to be stopped by the placenta, and we were unable to test our hypothesis. To bypass the tight placental microcirculation that limits crossing of the fetoplacental barrier (Firth and Leach 1996), we used diﬀusive gadolinium-DOTA. Gadolinium-DOTA can cross the placenta and be recovered in the embryonic bladder (Tanaka et al2001). It was used to assess mouse kidney function establishment in utero.

Materials and methods

Animals and anesthesia

Virgin female OF1 mice 9-11 weeks old were mated for 24 h (n=35) starting at 8:00 a.m. with male of the same genetic constitution. Because of the long mating period, it was impossible to accurately stage the embryos.

Therefore, embryonic stages presented in this paper took into account this 24-h conﬁdence interval and will al- ways be labeled as such. A fecundation rate of 43% was obtained. The 15 pregnant mice received a bolus of 37.5lmol of gadolinium-DOTA (Dotarem, Guerbet, France) via the catheterized tail vein using an 80-cm- long preﬁlled catheter (Folioplast, France) equipped with a 29-gauge needle (Terumo Europe, Belgium).

During MRI examination, mice were anesthetized by spontaneous inhalation of a mixture of isoﬂurane/oxy- gen (1.5–4%)/(1–1.5 l/min), and body temperature was controlled at 36–37C. Animal care was administered in strict accordance to the French Ministry of Agriculture regulations.

Magnetic resonance imaging

Experiments were performed on a Bruker Avance DRX 300 equipped with a vertical superwide-bore magnet operating at 7T and a shielded gradient system (maxi- mum gradient strength 144 mT/m, rising time <300 ls;

Bruker, Wissembourg, France). The resonant circuit of the MR probe was either a 38- or 64-mm diameter birdcage (Bruker, Rheinstetten, Germany) depending on mouse size. Mice were carefully fixed in the animal holder, then positioned vertically along the magnetic field direction. Sagittal, axial, and coronal scout images were performed to localize embryos, and imaging planes were defined in order to obtain pure sagittal or coronal virtual slicing of the targeted embryo. Images were then acquired using a field of view of 25·25 mm or 30·30 mm (depending on the mouse’s size) and a matrix 128·128 leading to an in-plane resolution of 198 or 234lm. If necessary, digital filtering was used in the

frequency-encoding direction to avoid aliasing. Five to nine 0.8–1-mm contiguous slices were acquired to cover the entire embryo.

T2-weighted images were acquired using an imaging method known as the multislice RARE method (Hen- ning et al. 1986). This method employs a single excita- tion step followed by the collection of multiple phase- encoded echoes. The repetition time (TR) was 2,000 ms and the echo time (TE) was 7.5 ms. A train of eight echoes was used to ﬁll thek-space, and the eﬀective TE was 31.7 ms. Four averages for each phase encoding were performed, resulting in a total acquisition time of 2 min 11 s.

Bruker ParaVision software was used for data acquisition and processing on a Silicon Graphics O2 workstation with the IRIX 6.5 operating system. Re- gions of interest were manually drawn over kidneys and bladder, and signal changes were expressed as a per- centage of initial signal intensity of the corresponding region of interest prior to contrast agent.

Results

Figure1shows a qualitative and quantitative evaluation of signal changes after gadolinium-DOTA injection in E14/15 pregnant mice. A preliminary experiment showed that a minimal dose of 35lmol of gadolinium–

DOTA is required to observe signal changes within the

Fig. 1 Sagittal T2-weighted magnetic resonance imagesaprior to andb30 min after injection of gadolinium-DOTA via the tail vein in a pregnant (E14/15) adult mouse. Note the signal drop within mother kidney and bladder as well as in placenta, those structures being localized in c. d Quantitative change in signal intensity in both structures, illustrating the ﬁltration process characterized by the delay between kidney (continuous line) and bladder (dotted line) signal variation. Data are presented as mean ± SEM and correspond to the gathering of three animals.

(3)

embryos. However, such a dose led to a negative contrast in the mother tissue, especially in kidney and bladder as well as in placenta (Fig. 1b). Quantitative analysis of signal changes is presented in Fig. 1d and reveals a 50% drop in signal intensity within 3 min for the kidney and within 9 min in the bladder.

Embryonic kidneys were unambiguously depicted without contrast agent injection in E15–16 embryos (Fig.2a). Nevertheless, injection of gadolinium-DOTA increased bladder and kidney signals and therefore im- proved the contrast between those organs and the sur- rounding structures (Fig. 2b). In younger embryos, gadolinium-DOTA may help characterize the kidneys, as they were observed in two out of the three E14–15 mice and were suspected in two out of the two E12–13 mice (Fig.3).

Kinetics of signal intensity changes in kidney and bladder were performed in seven out of the 15 mice included in the study. The bladder was observed in seven out of the nine E15–16 and older mice but in none of the earlier staged mice. Among those proﬁles, a delay was observed between the change in bladder signal compared with kidney signal in six animals and was similar in one of them (Fig. 2d).

Discussion

In utero imaging of developing embryos is challenging, especially with the massive development of genetically engineered mice for which early depiction of anatomical and functional modiﬁcations associated with gene mutation is of prime importance. Noninvasive MRI has proven to be an adapted tool to anatomically target submillimetric structures on both ﬁxed (Dhenain et al.

2001; Sugimoto et al.2002; Schneider et al.2003) and in utero mice embryos (Hogers et al. 2000; Chapon et al.

2002). However, from a biological point of view, a step forward must be made in order to address not only embryo anatomy but also tissue function establishment.

Ultrasound biomicroscopy-Doppler (Aristiza´bal et al.

1998; Linask and Huhta 2000; Phoon and Turnbull 2003) and micro-MRI pioneered this functional ap- proach by addressing quantitative cardiovascular ﬂuxes as well as cardiac function in mice and rat embryos (Smith2001; Hogers et al2000). However, none of these approaches was successfully applied to assess kidney function in utero. In the present study, we used gadolinium-DOTA, a diﬀusive contrast agent known to cross the placental barrier (Tanaka et al.2001), and followed the kinetics of renal clearance of this molecule from kidney to bladder.

Preliminary experiments showed that a limited quantity of gadolinium-DOTA crossed the placenta besides the injection of large doses of gadolinium- DOTA (‡35 lmol). Indeed, with such a signiﬁcant dose and with respect to the imaging parameters used, the T2 eﬀect of gadolinium-DOTA drives the contrast. The phenomenon is observed on the directly perfused tissue with a drop in signal, as shown in Fig.1, and especially on the mother kidney and bladder as well as on placenta.

The T1 enhancement in embryos (Figs.2b, 3b) reveals that only a limited quantity of gadolinium-DOTA crosses the placental barrier, as previously observed in rabbits (Novak et al.1993).

Usually, gadolinium-DOTA is associated with T1- weighted MRI but can also be used to modify contrast when T2-weighted MRI is performed (Fig.2b). More- over, at high magnetic ﬁeld, T2-weighted sequences produced anatomically relevant images (Smith et al.

1998; Hogers et al.2000; Chapon et al.2002) that allow unambiguous identiﬁcation of substructures such as kidneys and bladder within the embryo prior to contrast agent in about 2 min (Fig.2a).

Fig. 2 In utero saggital T2- weighted magnetic resonance (MR) images of an E15/16 embryoaprior to andb26 min after injection of gadolinium- DOTA via the mother tail vein.

cPlane where the images were performed.dKidney and bladder are identiﬁed.eKinetic data extracted from the serial of MR images acquired for kidney (continuous line) and bladder (dotted line). Data are presented as mean ± SEM and

correspond to the gathering of six animals.

(4)

To address the establishment of ﬁltration function, signal intensity changes in the kidney and bladder were measured. In adults, a delay between signal changes in kidney and bladder was observed, which can therefore be interpreted as the sign of ﬁltration function (Fig. 1d).

Both kinetics were performed only on seven out of the 15 mice included in the study. The bladder, which only reaches its deﬁnitive structure at the very end of gesta- tion (E17–18; Kaufman1992), was not seen in eight mice even after contrast agent injection. Indeed, no abdomi- nal enhancement that may correspond to the bladder was seen in six out of the six mice staged between E12–

13 and E14–15 and in two out of the nine E15–16 and older mice.

Kaufman’s histological study (Kaufman 1992) established that the bladder matures slowly and appears as a urogenital sinus up to embryonic stage 16–17. This may therefore account for the low detection rate of this structure. The fact that bladder was not observed in early staged mice does not allow us to conclude that the kidney is mature as soon as the diﬀerentiation between cortex and medulla starts. Indeed, as established by histology (Kaufman 1992), metanephros diﬀerentiation starts at embryonic stage 12–13 to lead to a clear dis- tinction between cortex and medulla at stage 14 and afterwards to an increase in kidney size up to the pups’

birth.

In older embryos, enhancement of the kidney and of the bladder was possible in seven cases out of nine, and a delay was observed in six of them (Fig. 2e), suggesting that glomerular function is established from E15–16.

A potential pitfall of this study deals with the enhancement of signal induced by gadolinium-DOTA.

Signal enhancement is dependent on the concentration of contrast agent (Rinck and Muller 1999). Even if it may be assumed that kidney volume at a deﬁned stage is similar from one mouse to another, it may not be the case for the bladder, of which the internal volume may vary. As a consequence and at constant ﬂux from kidneys to bladder, the latter may be more rapidly detected when its initial volume is reduced as the dilution of incoming gadolinium-DOTA is limited. One way to overcome this problem would be to measure the bladder volume, but, depending on its size and the resolution used for dynamic contrast imaging, the accuracy of the

measure may be disputed. However, because the delayed enhancement was observed between kidneys and bladder in six out of seven E15–16 and older mice, we may reasonably conclude that renal function is established in utero at this stage.

Conclusions

Dynamic contrast-enhanced MR microscopy is a unique tool that allows noninvasive evaluation of renal function in utero. The methodology used requires the detection of embryonic kidneys and bladder because the establishment of renal filtration is based on a delay in detection of gadolinium-DOTA between kidneys and bladder. The use of this diffusive contrast agent associated with T2- weighted images acquired in about 2 min with an in- plane resolution of 200lm emphasizes the establishment of glomerular filtration function at least at embryonic stage 15–16. Prior to this stage, because the bladder is not detected, the delayed enhancement strat- egy cannot be used.

AcknowledgmentsThe authors thank P. Legras and D. Gilbert for the housing and care of the animals used in this study.

References

Aristiza´bal O, Christopher DA, Foster FS, Turnbull DH (1998) 40- MHz echocardiography scanner for cardiovascular assessment of mouse embryos. Ultrasound Med Biol 24:1407–1417 Brune RM, Bard JBL, Dubreuil C, Guest E, Hill W, Kaufman M,

Stark M, Davidson D, Baldock RA (1999) A three-dimensional model of the mouse at embryonic day 9. Dev Biol 216:457–468 Chapon C, Franconi F, Roux J, Marescaux L, Le Jeune JJ, Lem- aire L (2002) In utero time-course assessment of mouse embryo development using high resolution magnetic resonance imaging.

Anat Embryol (Berl) 206(1–2):131–137

Congenital Malformations Australia (1981–1997). http://

www.npsu.unsw.edu.au/cm97.pdf

Dhenain M, Ruﬃns SW, Jacobs RE (2001) Three-dimensional digital mouse atlas using high-resolution MRI. Dev Biol 232(2):458–470

Firth JA, Leach L (1996) Not trophoblast alone: a review of the contribution of the fetal microvasculature to transplacental exchange. Placenta 17(2–3):89–96

Fig. 3 In utero saggital T2- weighted magnetic resonance images of an E12/13 embryoa prior to andb31 min after injection of gadolinium-DOTA via the mother tail vein. The arrowpoints to the suspected kidney.cPlane where the images were performed.

(5)

Grattan-Smith JD, Perez-Bayﬁeld MR, Jones RA, Little S, Broecker B, Smith EA, Scherz HC, Kirsch AJ (2003) MR imaging of kidneys: functional evaluation using F-15 perfusion imaging. Pediatric Radiol 33(5):293–304

Henning J, Nauerth A, Friedburg H (1986) RARE imaging; a fast imaging method for clinical MR. Magn Reson Med 3:823–833 Hogers B, Gross D, Lehmann V, Zick K, De Groot HJ, Gitten- berger-De Groot AC, Poelmann RE (2000) Magnetic resonance microscopy of mouse embryos in utero. Anat Rec 260(4):373–

377

Kaufman MH (1992) The atlas of mouse development. Academic Press, London

Linask KK, Huhta JC (2000) Use of Doppler echocardiography to monitor embryonic mouse heart function. Methods Mol Biol 135:245–252

Novak Z, Thurmond AS, Ross PL, Jones MK, Thornburg KL, Katzberg RW (1993) Gadolinium-DTPA transplacental trans- fer and distribution in fetal tissue in rabbits. Invest Radiol 28(9):828–830

Phoon CK, Turnbull DH (2003) Ultrasound biomicroscopy- Doppler in mouse cardiovascular development. Physiol Ge- nomics 24;14(1):3–15

Rinck PA, Muller RN (1999) Field strength and dose dependence of contrast enhancement by gadolinium-based MR contrast agents. Eur Radiol 9(5):998–1004

Schneider JE, Bamforth SD, Clarke K, Bhattacharya S, Neubauer S (2003) High-resolution, high-throughput magnetic resonance imaging of mouse embryonic anatomy using a fast gradient- echo sequence. MAGMA 16:43–51

Smith BR (2001) Magnetic resonance microscopy in cardiac development. Microsc Res Tech 52(3):323–330

Smith BR, Shattuck MD, Hedlund LW, Johnson GA (1998) Time- course imaging of rat embryos in utero with magnetic resonance microspcopy. Magn Reson Med 39:673–677

Sugimoto M, Manabe N, Morita M, Tanaka T, Okamoto R, Imanishi S, Miyamoto H (2002) Availability of NMR micro- scopic observation of mouse embryo disorder: examination in malformations induced by maternal administration of retinoic acid. J Vet Med Sci 64(5):427–433

Tanaka YO, Sohda S, Shigemitsu S, Niitsu M, Itai Y (2001) High temporal resolution dynamic contrast MRI in a high risk group for placenta accreta. Magn Reson Imaging 19(5):635–642 Turnbull DH, Bloomﬁeld TS, Baldwin HS, Foster FS, Joyner AL

(1995) Ultrasound backscatter microscope analysis of early mouse embryonic brain development. Proc Natl Acad Sci USA 92(6):2239–2243

Weintraub AS, Lin X, Itskovich VV, Aguinaldo JG, Chaplin WF, Denhardt DT, Fayad ZA (2004) Prenatal detection of embryo resorption in osteopontin-deﬁcient mice using serial noninvasive magnetic resonance microscopy. Pediatr Res 55(3):419–424