J. Biomedical Science and Engineering, 2011, 4, 315-319 JBiSE
doi:10.4236/jbise.2011.44041 Published Online April 2011 (http://www.SciRP.org/journal/jbise/).
Published Online April 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Renal functional MRI in mice evaluated with a dual bolus of
intravascular and diffusible contrast agents
Michael Pedersen1, Sukru Oguz Topcu2, Steven Sourbron3, Rikke Nørregaard2
1MR Research Center, University of Aarhus, Skejby, Denmark;
2Institute of Clinical Medicine, University of Aarhus, Skejby, Denmark;
3Division of Medical Physics, Leeds University Hospital, Leeds, Great Britain.
Received 15 February 2011; revised 12 March 2011; accepted 14 March 2011.
ABSTRACT
Background: The aim of this study was to evaluate
the feasibility of a dual-bolus protocol, where a first
bolus of an intravascular tracer is used to measure
perfusion, followed by a second bolus of a freely
filtered gadolinium-containing agent to measure fil-
tration capacity. Methods: The study was conducted
in mice subjected to complete unilateral ureteral
obstruction (UUO), and sham operated mice were
used as controls. Dynamic contrast- enhanced MRI
was performed 2 days after surgery. Results and
discussions: Mean signal-time curves of the renal
cortex, renal medulla and abdominal aorta were
used to calculate the relative renal blood flow ( rR BF ),
relative renal blood volume (rRBV), mean transit
time (MTT) and the glomerular transfer rate Ktrans.
We demonstrated that kidneys suffering from two
days of UUO showed a decrease in cortical as well
as medullary rRBF compared to kidneys from sham
-operated mice. Further, we found no changes in
rRBV and MTT among groups, neither in the cor-
tex nor in the medulla. The renal functional pa-
rameter Ktrans showed a tendency (but statistically
insignificant) to be reduced in the obstructed kid-
ney compared to the sham-operated mice. Conclu-
sions: We showed our first experiences with the
consecutive use of intra- and extravascularly dis-
tributed agents in a renal-diseased mouse model,
allowing analysis of both functional haemo-dyamics
and filtration capacity in kidneys.
Keywords: Renal Function; Dynamic Contrast-Enhanced
Magnetic Resonance Imaging; Intravascular Agents;
Extravascular Agents
1. INTRODUCTION
Ultrasmall superparamagnetic iron oxide particles (USPIO)
are small particles having a vascular remanence and are
taken up by the macrophages. Consequently, USPIOs
have a low clearance, facilitated mainly by the Kupffer
cells, resulting in a relatively large mean half-life (sev-
eral hours in humans). This pharmacokinetic behaviour
can also be used to reveal the intravascular characteris-
tics (blood perfusion) as they are usually not filtered
through the glomerular membrane [1,2]. In contrary, sma-
ller molecules like most gadolinium- containing agents
are freely filtered at the glomerulus and are neither ex-
creted nor reabsorbed, and these agents are therefore
suitable for measuring the filtration capacity in the kid-
ney [3]. The aim of this study was to evaluate the feasi-
bility of a dual-bolus protocol, where a first bolus of an
intravascular tracer is used to measure perfusion, fol-
lowed by a second bolus of a freely filtered gadolin-
ium-containing agent to measure filtration capacity. The
study was conducted in a renal impaired animal model
using mice subjected to complete unilateral ureteral ob-
struction, and sham operated mice were used as controls.
2. MATERIALS AND METHODS
2.1. Animal Handling
Studies were performed using adolescent mice (C57BL/6)
(~25 g). The animals were divided in two groups: UUO
group (n = 6) and sham operated group (n = 6). Each
animal was anesthetized with isoflurane (Abbott Scan-
dinavia, Solna, Sweden), and the mice were placed on a
heating pad during the operation to maintain rectal tem-
perature at 37˚C - 38˚C. The left ureter was exposed via
an abdominal transperitoneal incision using a micro-
scope with × 25 magnification and then completely oc-
cluded with a ligature. MRI was performed 2 days after
the surgical procedure. On the day of the imaging pro-
cedure, the mouse was reanaesthetized, and a polyure-
thane catheter was implanted in a vein tail for admini-
stration of drugs. All procedures conformed to the na-

M. Pedersen et al. / J. Biomedical Science and Engineering 4 (2011) 315-319 316
tional guidelines for the care and handling of animals
and the published guidelines from the National Institute
of Health.
2.2. Imaging Protocol
MRI was performed with a Philips Intera 1.5 T clinical
system (Philips Medical Systems, Best, The Netherlands)
equipped with actively shielded magnetic field gradients
with a maximal amplitude of 23 mT/m. RF excitation
was performed using the integrated body-coil, whereas a
surface coil with a diameter of 4 cm was employed for
reception. The animal was placed supine with the kid-
neys placed above the imaging coil. To avoid move-
ments of the kidneys during free breathing and magnetic
susceptibility artefacts related to the adjoining bowel, an
in-house-made plastic holder was used to block the
movements of the kidneys and isolate them from bowel
loops while avoiding compression of the parenchyma
and renal vessels. High-resolution T1-weighted images
were initially generated by a 3D gradient-echo pulse
sequence to allow accurate discrimination between cor-
tical and medullary components. Sequence parameters
were as follows: TR/TE = 1000 ms/100 ms, slice thick-
ness = 2 mm, field-of-view = 13 × 10 cm2, and acquisi-
tion and reconstruction matrix = 256 × 256. Number of
data averages was 16 to improve the signal-to-noise ratio.
A dynamic susceptibility-weighted gradient-echo sequ-
ence was applied during an iv injection of the an USPIO
(Sinarem; Guerbet, Paris, France), which was adminis-
tered rapidly in less than one second, corresponding to a
dose of 90 μmol Fe pr kg. A dynamic series of 120 im-
ages was acquired up to 120 s using the parameters:
repetition time (TR) = 50 ms, echo time (TE) = 20 ms
and flip-angle = 70˚, allowing images to be acquired
with an interval of 0.6 s. Acquired slice thickness was 2
mm and spatial resolution was 64 × 128, but was recon-
structed to a 256 × 256 matrix to allow overlaying on the
anatomical weighted image. Thirty minutes after injec-
tion of Sinerem, an iv bolus of 0.2 mmol/kg of a freely
diffusible gadolinium-containing agent (Magnevist; Bayer
Schering, Berlin, Germany) was administered in less
than one second. A dynamic T1-weighted gradient-echo
sequence was applied using the same orientation, spatial
resolution, number of slices, slice thickness, and voxel
size as before, and with the following parameters: imag-
ing time was set to 180 s and number of averages were
two to improve signal-to-noise ratio, TR = 12 ms, TE =
4 ms, and flip-angle = 9˚. All images were acquired in
the axial plane without respiratory compensation.
2.3. Image Analysis
Image analysis was performed using the Mistar software
package (Apollo Imaging Technology, Melbourne, Aus-
tralia). Region-of-interests (ROI) were drawn in the ab-
dominal aorta, renal (left) cortex and (left) medulla on
the high-resolution images, and ROIs were duplicated to
both the dynamic susceptibility-weighted and the dy-
namic contrast-enhanced images. Mean signal-time curves
of the three ROIs were computed. Dynamic susceptibil-
ity-weighted (Sinerem) data were analysed using the
standard model-free approach for intravascular contrast
agents [4], generating the parameters relative renal blood
flow (rRBF, in ml/100 ml/min), relative renal blood
volume (rRBV, in ml/100 ml), and mean transit time
(MTT, in s). Dynamic contrast-enhanced (Magnevist)
data were analysed assuming a linear relation between
signal change and tracer concentration, and using the
standard Tofts’ kinetic model [5], producing the transfer
constant (Ktrans, in 1/s) that can be a surrogate marker for
the permeability across the glomerular membrane (filtra-
tion). Statistical significance for the difference between
the two groups (obstructed and non-obstructed) was
evaluated with an unpaired Student’s t-test with equal
variance, and a unpaired t-test was applied to evaluate
statistical difference between contralateral (untouched)
kidneys in the UUO group and sham-operated kidneys.
Summarised data of rRBF, rRBV, MTT, and Ktrans are
presented as mean ± standard error of mean. The statis-
tical acceptance criterion was p < 0.05.
3. RESULTS
The dual bolus measurement was performed successfully
in all twelve mice. Examples of the time-concentration
curves following injection of Sinerem and Gd-DTPA,
respectively, are shown in Figure 1. Cortical rRBF in the
obstructed kidney was decreased compared to sham
cortical rRBF (227 ± 36 vs 317 ± 41 ml/min/100 ml; p =
0.02), and similarly, medullary rRBF in the obstructed
kidney was decreased compared to sham medullary
rRBF (60 ± 14 vs 100 ± 18 ml/min/100 ml; p = 0.007).
Cortical rRBV in the obstructed kidney was not signi-
ficantly changed compared to sham cortical rRBV (34 ±
9 vs 37 ± 7 ml/100 ml; p > 0.05), and obstructed
medullary rRBV was not significantly changed com-
pared to sham medullary rRBV (15 ± 2 vs 20 ± 3 ml/100
ml; p > 0.05). Obstructed cortical MTT was unchanged
compared to the sham group (9 ± 1 vs 7 ± 1 s; p > 0.05),
and similar findings were found in medulla (15 ± 2 vs 12
± 1 s; p > 0.05). Ktrans demonstrated no statistical
changes 48 h after obstruction compared to sham rats (9
± 2 vs 10 ± 1 s–1; p > 0.05). Data are presented in Figure
2. In neither case did parametric values differ among
contra- lateral (untouched) kidneys in the UUO group
and sham-operated kidneys (p > 0.05).
4. DISCUSSION
This experimental MRI study in mice demonstrated the
feasibility of a dual perfusion train, with a first injection

M. Pedersen et al. / J. Biomedical Science and Engineering 4 (2011) 315-319 317
(a) (b)
Figure 1. The relative signal intensity curves show the first of Sinerem (a) and Gd-DTPA (b) followed by continous
contrast material accumulation in the mouse kidney.
Figure 2. Parameters of cortical and medullary function measured in obstructed and sham mice: Relative renal blood flow
(rRBF), relative renal blood volume (rRBV), mean transit time (MTT) and the glomerular uptake transfer rate Ktrans.
of an intravascularly confined agent, and a second injec-
tion of an extravasating agent. Bolus-tracking data were
acquired with a dynamic susceptibility-weighted MRI
and a dynamic contrast-enhanced MRI sequence, respec-
tively. We demonstrated that kidneys suffering from two
days of UUO showed a decrease in cortical as well as
medullary renal blood flow (rRBF) compared to kidneys
from sham-operated mice. Further, we found no changes
in relative renal blood volume (rRBV) and MTT among
groups, neither in the cortex nor in the medulla. The re-
nal functional parameter Ktrans showed a tendency (but
statistically insignificant) to be reduced in the obstructed
kidney compared to the sham-operated mice.
It is well-known that complete obstruction of the uri-
nary tract has marked effects on RBF. In experimental
animals, complete unilateral ureteral obstruction (CUUO)
for 24 h causes a decrease in RBF in the obstructed kid-
ney [6-8]. Previous studies have demonstrated that, at 24
h into the obstruction, large areas of the cortical vascular
bed are either underperfused or not perfused at all [8,9].

M. Pedersen et al. / J. Biomedical Science and Engineering 4 (2011) 315-319 318
In addition, it has been observed in rats that RBF was
reduced to 30% of controls 6 days after CUUO [10].
Solez et al. also demonstrated a marked reduction in
inner medullary plasma flow (IMPF) in rats subjected to
CUUO for 18 h, whereas in the contralateral nonob-
structed kidney IMPF was not significantly different
from that in the control rats [11]. Our results are in
agreement with those observations. The calculated val-
ues of rRBF and rRBV are also in the order of magni-
tude of those recently reported in a pcy (polycystic kid-
neys and fibrosis) mouse model using a clinical 3 T MRI
system [12]. On the other hand, it is also known that
GFR is decreased in response to CUUO. After 24 h of
CUUO the continued fall in GFR of the obstructed kid-
ney is associated with a compensatory GFR increase in
the contralateral nonobstructed kidney [13]. Our study
could not confirm these findings. A possible explanation
for the lack of significant reduction in Ktrans (a surrogate
marker of GFR) may be an increase in glomerular filtra-
tion capacity at 48 h compared to the state at 24 h. An-
other explanation may be the use of mice in our study
instead of rats. A methodological explanation lies in the
known limitations of the Tofts’ model [5]. The parameter
Ktrans is often tacitly interpreted as measuring permeabil-
ity or filtration, but it is in fact a mixture of perfusion
and filtration. In certain regimes it may be interpreted as
perfusion (flow-limited), in others as filtration (perme-
ability-limited), but in most situations it will represent a
combination of both effects. This may well explain the
lack of specificity in our values for Ktrans, for instance if
the obstruction induces a transition from a permeability-
limited to a flow-limited regime, or vice versa. Moreover,
it has been shown in rabbits, humans and polycystic
mice [3,12,14] that the Tofts’ model does not provide an
accurate description of the kinetics of an MR tracer with
small molecular weight, measured at high temporal
resolution. For such data the model must be refined by
separating intra- and extravascular compartments [14].
This not only provides a more accurate description of the
data, but also a separate measurement of perfusion and
filtration, thereby eliminating any possible ambiguities
in the interpretation of Ktrans. The renophysiologial and
renofunctional changes following ureteral obstruction go
in parallel with volumetric changes (increased pelvis and
reduced cortex) and changes in mean cell density and
accumulation of myofibroblasts and collagen deposition
[15].
A second potential problem in the measurement of
GFR with T1-weighted bolus-tracking is the precise re-
lationship between signal change and tracer concentra-
tion. In this study, we assume a linear relationship be-
tween contrast agent concentration and measured signal
change. A related issue is that of limited water exchange
between intra- and extravascular space [16]. In the first
pass of a small molecular agent, strong concentration
differences exist between intra- and extravascular spaces.
This results in strong differences in T1 at peak concen-
tration between blood and tissue. The tissue then leaves
the fast-exchange regime, and a two-compartment model
for water kinetics must be used. Problems in the signal
analysis may also arise in the susceptibility-weighted
acquisition of the first bolus with Sinerem. A first prob-
lem is that the transverse relaxation rate 1/T2* in blood
is not linear with the iron concentration [17]. Moreover,
the results from the study by Bjørnerud et al indicated
that the presence of T1 effects can lead to a significant
underestimation of perfusion even in the absence of ex-
travascular leakage, as reflected in the area and peak
height of the first-pass curve following bolus injection of
iron oxide nanoparticles [17]. The renal concentration
time curves were deconvolved with the arterial input
function (AIF) in order to compensate for the dispersion
of the contrast bolus between the site of injection and the
tissue of interest, as well as the finite duration of the
bolus injection. The error estimated on deconvolved
perfusion data with T1 shortening has previously been
investigated by Aumann and coworkers [1], who dem-
onstrated that rRBV and rRBF were overestimated when
T1 shortening was not taken into account. Importantly, it
was notwithstanding stated that the error resulting from
this effect was negligible in the measurement of kidney
perfusion when using certain practical considerations
(dependent on sequence parameters and dose of the in-
travascular agent).
In conclusion, we showed our first experiences with
the consecutive use of intra- and extravascularly distrib-
uted agents in a renal-diseased mouse model, allowing
analysis of both functional haemodyamics and filtration
capacity in kidneys. The method was employed in mice
having a ureteral obstruction 48 h before MRI. These
promising findings encourage future use of this dual-
bolus approach in experimental settings, where non-
invasive determinations of renal perfusion and filtration
are requested.
5. ACKNOWLEDGEMENTS
We thank Guerbet for kindly providing vials of Sinerem.
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