Magnetic Resonance in Medicine 49:249 –257 (2003)
Renal and Systemic pH Imaging by Contrast-Enhanced MRI
Natarajan Raghunand,1* Christine Howison,1 A. Dean Sherry,2,3 Shanrong Zhang,2
and Robert J. Gillies1
Perturbations of renal and systemic pH accompany diseases of
the kidney, such as renal tubular acidosis, and the ability to
image tissue pH would be helpful to assess the extent and
severity of such conditions. A dual-contrast-agent strategy using two gadolinium agents, the pH-insensitive GdDOTP5ⴚ and
the pH-sensitive GdDOTA-4AmP5ⴚ, has been developed to generate pH maps by MRI. The renal pharmacokinetics of the
structurally dissimilar pH-insensitive contrast agents GdDTPA2ⴚ and GdDOTP5ⴚ were found to be similar. On that basis,
and on the basis of similarity of structure and charge, the renal
pharmacokinetics of GdDOTP5ⴚ and GdDOTA-4AmP5ⴚ were
assumed to be identical. Dynamic T1-weighted images of mice
were acquired for 1 hr each following boluses of GdDOTP5ⴚ and
GdDOTA-4AmP5ⴚ. The time-varying apparent concentration of
GdDOTP5ⴚ and the time-varying enhancement in longitudinal
relaxation rate following GdDOTA-4AmP5ⴚ were calculated for
each pixel and used to compute pH images of the kidneys and
surrounding tissues. MRI pH maps of control mice show acidic
regions corresponding to the renal papilla, calyx, and ureter.
Pretreatment of mice with the carbonic anhydrase inhibitor
acetazolamide resulted in systemic metabolic acidosis and accompanying urine alkalinization that was readily detected by
this dual-contrast-agent approach.
Magn Reson Med 49:
249 –257, 2003. © 2003 Wiley-Liss, Inc.
Key words: gadolinium; kidney; relaxivity; acetazolamide; acidbase
The pH of bodily fluids affects the organism in many ways,
especially through its effects on cellular and plasma proteins. Maintenance of acid-base homeostasis is critical,
and occurs at several levels. The most immediate and local
response to an acid or alkali load is through chemical
processes, including intracellular, extracellular, and bone
buffers. Buffering capacity is limited, and active and passive physiologic responses are also required to maintain
the acid-base balance. These physiologic processes can be
at the cellular level, such as through feedback changes in
metabolism, and at the systemic level, involving adaptive
changes to the excretion of volatile acids by the lungs and
fixed acids by the kidneys (1). In most tissues the buffering
1
Cancer Center Division, University of Arizona Health Sciences Center, Tucson, Arizona.
2
Department of Chemistry, University of Texas at Dallas, Richardson, Texas.
3
Rogers Magnetic Resonance Center, University of Texas Southwestern
Medical Center, Dallas, Texas.
Grant sponsor: American Cancer Society; Grant number: IRG7400124451224; Grant sponsor: NIH; Grant numbers: R21-CA84697; R01-CA77575;
R24-CA83148.
Presented in part at the meeting of Contrast Media Research, Capri, Italy,
2001.
*Correspondence to: Natarajan Raghunand, University of Arizona Cancer
Center, 1515 N. Campbell Ave., Tucson, AZ 85724-5024.
E-mail: [email protected]
Received 25 February 2002; revised 9 September 2002; accepted
12 September 2002.
DOI 10.1002/mrm.10347
Published online in Wiley InterScience (www.interscience.wiley.com).
© 2003 Wiley-Liss, Inc.
capacities of intra- and extracellular fluid are roughly
equal, but the approximately 1.5:1 greater volume of intracellular fluid compared to extracellular fluid results in a
greater contribution of intracellular buffers to overall buffering in these tissues (2).
Certain pathologies are associated with a perturbed pH
homeostasis. For example, tumor interstitial fluid has a
reduced buffering capacity compared to normal tissue, and
this, in combination with poor perfusion and increased
lactic acid secretion by tumors (3), is believed to result in
an acidic extracellular pH in tumors (4). This situation is
exacerbated by the lowered buffering capacity of bicarbonate at more acid pH. This acidic pH of tumors has numerous consequences, including resistance to radio- and chemotherapies (5). Pathologically altered renal physiology
can also manifest with perturbations in both systemic and
renal pH. Hereditary defects and acquired deficiencies in
renal tubular ion transport systems can result in systemic
metabolic acidosis or alkalosis (6,7). Therapies to correct
these conditions include the use of alkalinizing or acidifying buffers. Novel gene therapies have also shown promise for the treatment of some of these diseases, but there are
still problems with heterogeneous gene delivery to the
target tissue, and loss of corrective genes from that tissue
over time (8,9). Methodologies to image the spatial distribution of tissue pH would have considerable biomedical
and clinical relevance in such cases because they would
enable the noninvasive assessment of disease extent, progression, and response to therapy.
Several methods have been proposed to measure tissue
pH by MRS and MRI. Some exploit endogenous MR resonances, while others require the administration of exogenous agents. Intracellular pH in tissues can be estimated
from the 31P MR resonance of inorganic phosphate (Pi) (3).
The pH-sensitive 31P MR resonance of 3-aminopropylphosphate (3-APP) has been used by us and other researchers to measure extracellular pH of tumors and normal tissues in mice (4,5,10). Molecules with pH-sensitive
19
F and 1H resonances have also been reported. Ojugo et al.
(10) reported that pH measurements with excellent signalto-noise ratio (SNR) per time period are possible with the
extracellular 19F-containing pH probe, ZK-150471. They
list the lack of potentially interfering endogenous 19F resonances, and the higher NMR and pH sensitivity of ZK150471 as advantages of the 19F probe compared to 3-APP.
Both 3-APP and ZK-150471 are cell-impermeant and report only the extracellular pH, although 31P MRS of 3-APP
offers the possibility of simultaneous measurement of intracellular pH from the Pi resonance. Mason and coworkers (11) reported that a fluorinated derivative of vitamin
B6, 6-fluoropyridoxol, readily enters cells, and they used
this single molecule to measure the intra- and extracellular
pH in rodent tumors by 19F MRS.
249
250
The 1H nucleus offers the highest inherent sensitivity,
and it is possible to image the spatial distribution of tissue
pH in vivo by the use of probes with pH-sensitive 1H
resonances. We and others have employed an exogenously
administered imidazole, IEPA, for imaging breast (12) and
brain (13) tumor pH in rodents by MRS imaging (MRSI) of
the pH-sensitive resonance of the protons on the C-2 carbon in the imidazole ring. This resonance is in the
8 –9 ppm region, with few interfering resonances. Endogenous imidazoles, such as histidine, also have pH-sensitive 1H resonances in this region, but their concentrations
are too low to exploit. However, Vermathen et al. (14)
successfully measured brain pH in humans by localized
1
H MRS after oral administration of histidine. MRS and
MRSI techniques for measuring pH suffer a disadvantage
in sensitivity, and therefore spatial resolution, but have
the advantage that the measurement is independent of the
concentration of the pH probe.
A different approach to exploiting endogenous resonances to measure tissue pH has been proposed by Van
Zijl and colleagues (15). They point out that the region of
the 1H MR spectrum ⬎ 5 ppm contains several very low
and undetectable resonances from NH protons that are
typically in fast exchange with solvent water. This exchange process renders these resonances nearly impossible to detect with pulse sequences that include a water
presaturation pulse. However, using a pulse sequence that
permits visualization of these resonances, Van Zijl et al.
(15) demonstrated that it is possible to measure pH from
the pH-sensitive rate of exchange with water protons of
one or more of these protons. Ward and Balaban (16)
demonstrated that a compound (such as 5,6-dihydrouracil)
with multiple proton exchange sites, each with different
pH dependencies, may be used to measure pH using a
“ratiometric” method.
pH-sensitive gadolinium complexes (17,18) and gadolinium-containing pH-sensitive polyion complexes (19) offer the possibility of imaging pH with a spatial resolution
comparable to standard MRI. However, the enhancement
observed in an image will be dependent on not only the
pH, but also the local concentration of the agent. One
approach to determine pH is to sequentially administer
two contrast agents with identical tissue pharmacokinetics, one being insensitive to tissue pH and the other being
pH-sensitive (20). The distribution of the pH-insensitive
agent can be used to predict the concentration of the pHsensitive agent. Recently a gadolinium-based pH-sensitive
contrast agent, GdDOTA-4AmP5⫺, was introduced by
Sherry and coworkers (17). The H-bonding network created by protonation of the phosphonates on the side-arms
of this complex provides a catalytic pathway for exchange
of the bound water protons with protons of bulk water,
making the longitudinal water proton relaxivity (r1) of this
molecule pH-sensitive. In the present work we demonstrate computed pH images of kidneys and nearby tissues
in mice following sequential injections of GdDOTA4AmP5⫺ and the pH-insensitive tetraphosphonate analog,
GdDOTP5⫺. The pharmacokinetics of the pH-insensitive
agent were monitored prior to and following injection of
the GdDOTA-4AmP5⫺, to alert us to changes in animal
physiology during the course of the experiment. When the
pharmacokinetics of the two GdDOTP5⫺ boluses com-
Raghunand et al.
pared favorably, we reasoned that the intervening GdDOTA-4AmP5⫺ would have followed the same time-dependent distribution.
METHODS
In Vivo MRI Experiments
All MRI experiments were performed on a Bruker Biospec® 4.7 T system with 14 G/cm self-shielded gradients.
Female SCID and C3H mice weighing 19 –26 g were imaged using a 25-mm-ID Helmholtz coil (Doty Scientific,
Columbia, SC). Mice were anesthetized by inhaled isoflurane (1.5%, rest O2 at 1 L/min), and cannulated at the
tail-vein. To minimize breathing-related motion, the animals were taped down gently into a holder, which was
then positioned inside the coil. Temperature was monitored during all in vivo MRI experiments using a rectal
fluoroptic temperature probe (Luxtron Corporation, Santa
Clara, CA), and was maintained at 37–37.5°C by a recirculating warm-water blanket. Two contrast agents were used
in any given animal: GdDOTP5⫺ (Macrocyclics, Dallas,
TX), and either GdDOTA-4AmP5⫺ (Macrocyclics) or GdDTPA2⫺ (Magnevist®; Berlex, Fairfield, NJ). The structures
and molecular weights of these contrast agents are shown
in Fig. 1. The tail-vein cannula was capped with a heparinized septum to permit repeated injections. The dead
volume of the infusion line was approximately 0.1 mL.
Contrast agent (0.025– 0.1 mmole/Kg, as a 25-mM or
50-mM solution) was administered and chased with
0.15 mL heparinized saline over 40 s via this catheter at
the appropriate time during each experiment. The slow
rate of injection was used partly to allow the animal to
tolerate the hypervolemic bolus, and partly because the
reproducibility of slow injections was better than that of
rapid boluses.
Scout images were collected to plan coronal slices
through the kidneys of the mouse. Once the slice scheme
was finalized, a fat-suppressed proton-density spin-echo
(SE) image was obtained with the following parameters:
recycle time ⫽ 8 s, echo time (TE) ⫽ 6 ms, number of
averages ⫽ 2, field of view (FOV) ⫽ 3.5 cm, matrix ⫽ 128 ⫻
128, slice thickness ⫽ 1–1.3 mm, and number of slices ⫽
2. The contrast-enhanced portion of each pH imaging experiment consisted of three consecutive phases: fat-suppressed SE images were acquired every 30 – 40 s for 1 hr
following 1) a bolus of GdDOTP5⫺, 2) a bolus of GdDOTA4AmP5⫺, and 3) a bolus of GdDOTP5⫺, with the following
imaging parameters: recycle time ⫽ 80 ms, TE ⫽ 6 ms,
number of averages ⫽ 3– 4 (other parameters as given
above for the proton-density image). Tissue pH maps were
calculated for three mice from the pharmacokinetics of
GdDOTP5⫺ and GdDOTA-4AmP5⫺ and the pH-dependent
relaxivity of GdDOTA-4AmP5⫺ measured in vitro at 37°C
and 4.7 T. In separate experiments, the renal pharmacokinetics of the two pH-insensitive molecules GdDOTP5⫺ and
GdDTPA2⫺ were compared in four mice.
Acetazolamide is a carbonic anhydrase inhibitor that
causes metabolic acidosis and concomitant urinary alkalinization by inhibiting resorption of filtered HCO3⫺ (21).
Mice were administered 30 mg/Kg/day acetazolamide
(Sigma, St. Louis, MO) once a day orally for 7 days prior to
pH Imaging Using GdDOTA-4AmP5⫺
251
FIG. 1. a: GdDTPA2⫺, mol. wt. 545.6, assuming no protonation at pH 7.4. b: GdDOTP5⫺, mol. wt. 699.5, assuming 2 H⫹ bound at pH 7.4. c:
GdDOTA-4AmP5⫺, mol. wt. 927.7, assuming 2 H⫹ bound at pH 7.4.
imaging (21,22). The dose on the day of the imaging experiment was administered 1 hr prior to induction of anesthesia in the animal. All experiments were carried out in
compliance with the guidelines of the University of Arizona Institutional Animal Care and Use Committee. All
data were processed using programs written in-house in
Interactive Data Language (IDL; Research Systems, Boulder, CO).
In Vitro Relaxivity Measurements
GdDTPA2⫺ and GdDOTP5⫺ solutions were made in
10 different concentrations of 0 –5 mM in phosphate-buffered saline (PBS), pH 7, loaded in 2-mL microcentrifuge
tubes, and the water-proton T1 values were measured at
37°C and 4.7 T. Similarly, the water-proton relaxivity of
GdDOTA-4AmP5⫺ was measured in PBS at several pH
values between 5.75– 8.0. T1 measurements were made
using a 12-segment, high-pass, 70-mm-ID birdcage coil.
Sample temperature, maintained at 37°C by blowing warm
air through glass wool surrounding the sample tray, was
monitored using a fluoroptic temperature probe. Longitudinal relaxation times were measured by SE imaging using
20 recovery times of 40 – 8000 ms, with a TE of 6 ms. Small
molecules such as these are excreted by glomerular filtration, and most of the contrast agent detected in kidney by
MRI is expected to reside in the postglomerular filtrate.
The filtrate normally has a low protein content due to the
low permeability of the glomerular capillary wall to large
molecules (23). Thus, PBS rather than serum or other
protein-containing solution was chosen as the medium for
determination of in vitro relaxivities.
isolated compartments: 1) the filtrate, containing a high
concentration of the contrast agent; and 2) reabsorbed water, containing essentially no magnetopharmaceutical. If
water exchange between these two compartments is restricted, it would explain the reduced apparent relaxivity
of GdDTPA2⫺ observed in the kidney in vivo. Landis et al.
(26) demonstrated that the assumption of fast water exchange between intra- and extracellular compartments is
invalid in rat muscle for concentrations of GdDTPA2⫺
greater than approximately 0.1 mM. This leads to an underestimation of the extracellular concentration of the contrast agent in typical dynamic MRI experiments, when
relaxivities measured in vitro in saline or other cell-free
solutions are directly applied to in vivo signal enhancement data.
Consider the case in which water proton exchange between the reabsorbed water (compartment A) and filtrate
(compartment B) in the kidney is near the slow-exchange
limit. The Boltzmann equilibrium magnetizations (or proton-density signal intensities) in the two compartments are
SA and SB, the corresponding precontrast longitudinal relaxation times are TA and TB, and the recycle time for SE
imaging is TR. Neglecting T2-related signal decay (TE ⫽
6 ms), and also assuming fast water mixing within each
compartment, the net instantaneous magnetization (or precontrast signal intensity) can be written as:
S 1 ⫽ S A 关1 ⫺ exp共 ⫺ TR /TA 兲兴 ⫹ SB 关1 ⫺ exp共 ⫺ TR /TB 兲兴.
[1]
Provided TR ⬍⬍ TA and TR ⬍⬍ TB, the exponential terms in
the above equation can be approximated with Taylor series
expansions to only the first-order terms:
Effects of Compartmentation and Slow Water Exchange
Tissue structure and physiology can alter the in vivo relaxivities of contrast agents. For example, GdDTPA2⫺ relaxivities in rat kidneys in vivo are substantially lower in
the cortex and medulla as compared to in vitro relaxivities
measured in saline, and to in vivo relaxivities measured in
other tissues (24,25). This is possibly due to compartmentalization of the contrast agent molecules in the kidney
and slow rates of water exchange between the compartments. Shuter et al. (24) noted that the kidneys create two
S 1 ⬇ S A 䡠 T R/T A ⫹ S B 䡠 T R/T B.
[2]
After administration of GdDOTP5⫺, assuming that the contrast agent is only in compartment B, the postcontrast
signal intensity can be approximated as:
S 2 ⬇ S A 䡠 T R/T A ⫹ S B 䡠 T R
⫻ 关1 ⫹ r DOTP 䡠 T B 䡠 关GdDOTP 5⫺兴兴/T B
[3]
252
Raghunand et al.
where rDOTP is the longitudinal relaxivity of GdDOTP5⫺ in
compartment B, which we assume to be the same as that
measured in vitro in PBS. The above Taylor series approximation to the first-order terms only holds for regions in
which TR is much less than the postcontrast longitudinal
relaxation time TB/(1 ⫹ rDOTP 䡠 TB 䡠 [GdDOTP5⫺]), and may
not be valid in regions with very high concentrations of
contrast agent (such as the ureters). A short recycle time of
80 ms was used in order to maximize the validity of this
approximation. Combining Eqs. [2] and [3] yields:
关GdDOTP 5⫺兴 ⬇ 共S 2 ⫺ S 1兲/共S B 䡠 T R 䡠 r DOTP兲.
[4]
As the compartment-specific SB cannot be measured, we
have instead used (SA ⫹ SB), the sum of the magnetization
in both compartments, obtained from the precontrast proton-density image. Thus, compartmentation of the contrast
agent results in an underestimation of the concentration of
the agent in compartment B by the factor SB/(SA ⫹ SB). It
must be noted that in these experiments S2 and [GdDOTP5⫺] are functions of time, as the agent is cleared from
the animal.
One hour after administration of GdDOTP5⫺ an identical
bolus of GdDOTA-4AmP5⫺ was administered to the animal. S3 represents the time-varying signal intensity following administration of GdDOTA-4AmP5⫺, and includes enhancement due to both residual GdDOTP5⫺ and fresh GdDOTA-4AmP5⫺. The combined concentrations of residual
GdDOTP5⫺ and fresh GdDOTA-4AmP5⫺ are assumed to
remain within the linear regime for dependence of signal
enhancement on the concentrations of both agents. If the
solution milieu in the filtrate (compartment B) is similar to
saline, then it is worth noting that the signal enhancement
provided by GdDOTP5⫺ and GdDOTA-4AmP5⫺ in PBS
was linear up to 4 mM with our SE imaging parameters
(vide infra). The concentration of contrast agent probably
does not exceed 4 mM in the cortex or medulla for any
significant length of time. However, it is probably in excess
of 4 mM in the ureters (and bladder), as it gets concentrated at longer times postinjection. For the other regions
the following expression can be written for the observed
relaxivity of GdDOTA-4AmP5⫺, r4AmP:
r 4AmP ⬇
S3 ⫺ S1
S B 䡠 T R 䡠 关GdDOTA ⫺ 4AmP 5⫺兴
⫺ r DOTP 䡠
⬇
关GdDOTP 5⫺,residual兴
关GdDOTA ⫺ 4AmP 5⫺兴
from the data collected 30 – 60 min post-GdDOTP5⫺, and
extrapolated for calculating [GdDOTP5⫺, residual] in each
pixel at each time-point post-GdDOTA-4AmP5⫺. Let us
consider the two terms on the right side of Eq. [5]. The
underestimation of [GdDOTP5⫺] by use of (SA ⫹ SB) in
place of SB in Eq. [4] results in overestimation of the first
term. But we have used (SA ⫹ SB) in place of SB here too,
and this cancels out the error in the first term. In the
second term, both [GdDOTP5⫺, residual] in the numerator
and [GdDOTP5⫺] in the denominator are underestimated
by the same factor, and these errors cancel out as well.
Thus, to a first approximation, we can use this dual-contrast-agent strategy in conjunction with T1-weighted imaging to compute the in vivo longitudinal water proton relaxivity of GdDOTA-4AmP5⫺, even in the presence of
compartmentation of contrast agent and slow water exchange between the compartments.
RESULTS AND DISCUSSION
In Vitro Calibrations
The longitudinal water proton relaxivity r1 of each contrast agent was calculated from a fit to the following equation:
1/T 1 ⫽ 1/T 10 ⫹ r 1 • 关Gd兴
where [Gd] is the concentration of the contrast agent, and
T10 and T1 are the longitudinal relaxation times of water
protons in the absence and presence of contrast agent,
respectively. The relaxivity of GdDTPA2⫺ in PBS at 37°C
and 4.7 T was measured to be 3.2 ⫾ 0.27 mM⫺1s⫺1, which
is in good agreement (mutatis mutandis) with the values
reported by Landis et al. (26) for GdDTPA2⫺ in saline at
37°C at 4.0 T and by Rozijn et al. (27) for GdDTPA2⫺ in
water at 37°C and 6.3 T. The relaxivity of GdDOTP5⫺ in
PBS at 37°C and 4.7 T was 3.0 ⫾ 0.34 mM⫺1s⫺1. Aime et al.
(28) reported a value of ⬇3.3 mM⫺1s⫺1 for GdDOTP5⫺ at
60 MHz and 35°C. For GdDTPA2⫺, GdDOTP5⫺, and GdDOTA-4AmP5⫺ at all the pH values tested, signal enhancement (with TE ⫽ 6 ms) varied linearly with concentration
up to 4 mM (r2 ⬎ 0.97 in all cases). Figure 2 shows the
pH-dependent variation in water proton relaxivity (r4AmP)
of GdDOTA-4AmP5⫺ at 37°C and 4.7 T. The data between
pH 5.75– 8.0 can be described by the following polynomial
function (r2 ⬎ 0.99):
pH ⫽ 1.62 ⫹ 4.50•共r 4AmP兲 ⫺ 0.78•共r 4AmP兲 2.
关GdDOTP 5⫺,residual兴
S3 ⫺ S1
.
5⫺ ⫺ r DOTP 䡠
S B 䡠 T R 䡠 关GdDOTP 兴
关GdDOTP 5⫺兴
[5]
The second portion of Eq. [5] depicts our assumption that
the pharmacokinetics of GdDOTA-4AmP5⫺ will be essentially identical to that of GdDOTP5⫺, given their similar
molecular structure and charge. S3 and [GdDOTP5⫺] are
functions of time in this dynamic experiment, and the
above equations are written for a given time-point. A biexponential function approximating the time-dependent decay of signal intensity was fitted on a pixel-by-pixel basis
[6]
[7]
There is an inflection point near pH 5.75, and this equation
is only applicable for computing the pH in samples within
the pH range 5.75– 8.0. Unambiguous assignment of pH to
a measured in vivo relaxivity can be made only in samples
with pH values that fall strictly within any one monotonic
region of the curve. For example, in our calculations a
region at pH 5.25 would be misassigned to ⬇pH 6.48 on
the other side of the inflection point.
Renal Pharmacokinetics: GdDTPA2⫺ vs. GdDOTP5⫺
We compared the renal pharmacokinetics of GdDTPA2⫺
and GdDOTP5⫺ in four mice. The data from one animal are
pH Imaging Using GdDOTA-4AmP5⫺
FIG. 2. The pH dependence of the water-proton longitudinal relaxivity of GdDOTA-4AmP5⫺ (r4AmP) in PBS at 37°C, 4.7 T. The solid line
represents a curve-fit using a second-order polynomial function. In
in vivo experiments pH was back-calculated from the calculated in
vivo relaxivity of GdDOTA-4AmP5⫺ using the fitted relation pH ⫽
1.62 ⫹ 4.5(r4AmP) – 0.78 (r4AmP)2 (r2 ⬎ 0.99).
253
shown in Fig. 3. T1-weighted SE images were collected
dynamically for 1 hr following a bolus of GdDTPA2⫺
(0.05 mmole/Kg) using the parameters described above. A
bolus of GdDOTP5⫺ (0.05 mmole/Kg) was then administered and images were collected for another hour. Figure
3a depicts the apparent concentration of GdDTPA2⫺ calculated at each time-point from three regions-of-interest
(ROIs) in the kidneys, while Fig. 3b shows the apparent
concentration of GdDOTP5⫺ calculated at each time-point
for the same ROIs. As explained above, residual enhancement from the first (GdDTPA2⫺) bolus was estimated by
extrapolation using a biexponential fitted function, and
subtracted out prior to calculation of the apparent concentration of GdDOTP5⫺ at each time-point following the
second bolus.
The pharmacokinetics of GdDTPA2⫺ and GdDOTP5⫺
appear to be roughly comparable in the renal cortex and
medulla, within the noise in the data in Fig. 3a and b.
Some divergence in the apparent pharmacokinetics of the
two molecules is visible, especially at the early timepoints, in the calyx-ureter ROI. The ROI data in Fig. 3a and
b have an unavoidable component of noise arising from
physiologic variability. The noise is more severe when the
FIG. 3. Representative comparison of the renal pharmacokinetics of GdDTPA2⫺ and GdDOTP5⫺ in a female C3H/hen mouse. Data are
averages from ROIs drawn by hand over the cortex (solid line), medulla (dotted), and calyx-ureter (dashed) in both kidneys and both imaging
slices. Images were acquired for 1 hr each following first a bolus of GdDTPA2⫺ (0.05 mmole/Kg) and then a bolus of GdDOTP5⫺
(0.05 mmole/Kg). a and c: Data acquired following the GdDTPA2⫺ bolus. b and d: Data collected following the GdDOTP5⫺ bolus. The
ordinate in panels a and b is the apparent concentration of the contrast agent calculated at each time-point. The ordinate in panels c and
d is the AUC/time concentration calculated as per Eq. [8]. The signal enhancement following the GdDTPA2⫺ bolus was converted to
concentration by using r1 ⫽ 3.2 mM⫺1 s⫺1. Residual enhancement from the GdDTPA2⫺ bolus was estimated by extrapolation and
subtracted prior to calculation of the apparent concentration of GdDOTP5⫺ following the second bolus.
254
Raghunand et al.
Table 1
Renal Pharmacokinetics of GdDTPA2⫺ and GdDOTP5⫺
ROI
C3H/
hena
Cortex
Medulla
Ureter
C3H/
hena
Cortex
Medulla
Ureter
SCIDa
Cortex
Medulla
Ureter
SCIDa
Cortex
Medulla
Ureter
AUC/time at
30 min, mM
1st bolus
AUC/time at
30 min, mM
2nd bolus
Ratio
bolus 2/bolus1
0.41
0.88
1.28
0.37
0.80
1.05
0.90
0.91
0.82
0.89
1.25
1.63
0.95
1.32
1.51
1.07
1.06
0.93
0.32
0.58
0.87
0.29
0.53
0.72
0.91
0.91
0.83
0.41
0.56
0.86
0.44
0.62
0.79
1.07
1.11
0.92
Renal Pharmacokinetics: GdDOTP5⫺ vs.
GdDOTA-4AmP5⫺
a
Bolus sequence for the C3H/hen mice was GdDTPA2⫺ followed by
GdDOTP5⫺, and vice-versa for the SCID mice.
data are examined on a pixel-by-pixel basis, making reliable computations of pharmacokinetics from these data
challenging. Another complication is that medullary pixels contain both proximal and distal tubules, with the
concentration of the contrast agent varying along the
length of the nephron. To reduce these artifacts, an areaunder-the-curve approach was used to smooth the timevarying contrast agent concentration, C(t), in each pixel.
The area-under-the-curve (AUC) for each pixel at each
time-point was divided by the time postinjection (t) to
yield AUC/time:
AUC/time ⫽
冕
t
C共t兲dt
0
t
.
0.87 ⫾ 0.05 in the calyx-ureter ROI (Table 1). The apparent
AUC/time concentration of contrast agent in the calyxureter ROI was always higher 30 min following the first
bolus of contrast agent than 30 min following the second
bolus. The average concentration of contrast agent is
higher in the calyx and ureters than in other regions of the
kidney. It is possible that high total (residual ⫹ fresh)
contrast agent concentration in the calyx-ureter ROI following the second bolus results in a significant shortening
of T2 in these regions, with a reduction in the signal
enhancement in our T1-weighted SE images. This would
lead to the computation of a decreased apparent concentration of contrast agent following the second bolus.
Hence, the pharmacokinetics were highly consistent in the
cortex and medulla, and less so in the calyx-ureter.
[8]
Implicit in this manipulation is the assumption that signal
enhancement maintains a linear dependence on contrast
agent concentration in all pixels at all times. As discussed
above, this assumption may fail in regions with very high
contrast agent concentrations, such as the calyx and ureters.
The data in Fig. 3a and b are replotted in Fig. 3c and d as
AUC/time vs. time postinjection. In this example, agreement between the two pharmacokinetic curves was within
10% at 30 min postinjection in the cortex (0.89 mM vs.
0.95 mM), the medulla (1.25 mM vs. 1.32 mM), and the
calyx-ureter (1.63 mM vs. 1.51 mM). Data from two different strains of mice, C3H/hen and SCID, are shown in Table
1. The sequence of contrast agent administration was GdDTPA2⫺ followed by GdDOTP5⫺ in the C3H mice, and
vice versa in the SCID mice. Over all mice, the ratios of the
AUC/time concentration of contrast agent at 30 min
postinjection of the second bolus to the first bolus were
0.99 ⫾ 0.08 in the cortex, 1.0 ⫾ 0.09 in the medulla, and
GdDOTP5⫺ and GdDOTA-4AmP5⫺ are arguably more similar to one another than are GdDTPA2⫺ and GdDOTP5⫺
(Fig. 1). Therefore, given the similarity in renal pharmacokinetics of the latter pair of molecules (Fig. 3 and Table 1),
we made the assumption that the MRI pharmacokinetics of
GdDOTP5⫺ is an adequate surrogate for the pharmacokinetics of GdDOTA-4AmP5⫺ in a given mouse. We also
compared the uptake kinetics of GdDOTP5⫺ and GdDOTA4AmP5⫺, as well as the reproducibility of pharmacokinetics following two identical boluses of GdDOTP5⫺, in three
mice. T1-weighted SE images were collected dynamically
for 1 hr each following first a bolus of GdDOTP5⫺
(0.05 mmole/Kg), then a bolus of GdDOTA4-AmP5⫺
(0.05 mmole/Kg), and finally a second bolus of GdDOTP5⫺
(0.05 mmole/Kg).
Representative data from one experiment are shown in
Fig. 4a– c. The AUC/time apparent concentrations of GdDOTP5⫺ in three ROIs following the first and last bolus are
plotted in Fig. 4a and c, respectively. The AUC/time enhancement in apparent relaxation rate following the middle bolus of GdDOTA-4AmP5⫺ is plotted for the same
three ROIs in Fig. 4b. In the data plotted in Fig. 4b and c,
residual enhancement from previous bolus(es) was accounted for as explained above. In Fig. 4a and c we compare the reproducibility of the pharmacokinetics of GdDOTP5⫺ in an animal. In this example, agreement between
the two boluses was good at 30 min postinjection in the
cortex (0.80 mM vs. 0.72 mM) and the medulla (0.89 mM
vs. 0.98 mM), but not in the calyx-ureter (2.66 mM vs.
2.27 mM).
Since the abscissa in Fig. 4b is different from those in
Fig. 4a and c, we compared the pharmacokinetics of the
three boluses on the basis of “time to 90% of maximum
AUC/time intensity” (t90). In Fig. 4a– c, respectively, the
t90 is 7.3/7.6/8.6 min in the cortex, 9.0/9.7/9.3 min in the
medulla, and 9.3/10.6/10.0 min in the calyx-ureter. Agreement between the three boluses on the basis of t90 was
good in all three ROIs in three out of three mice. The
standard deviation (SD) in the t90 of the three boluses,
when expressed as percent of the mean, was within 7.1%
in all ROIs in all three mice (Table 2). These results
strengthen our assumption that GdDOTP5⫺ and GdDOTA4AmP5⫺ exhibit similar kinetics of renal uptake, and also
pH Imaging Using GdDOTA-4AmP5⫺
255
FIG. 4. Representative comparison of the renal pharmacokinetics of GdDOTP5⫺ and GdDOTA-4AmP5⫺ in a female SCID mouse. Images
were acquired for 1 hr each following first a bolus of GdDOTP5⫺ (0.05 mmole/Kg), then a bolus of GdDOTA-4AmP5⫺ (0.05 mmole/Kg), and
then a second bolus of GdDOTP5⫺ (0.05 mmole/Kg). Shown are the data obtained following (a) the first bolus of GdDOTP5⫺, (b) the bolus
of GdDOTA-4AmP5⫺, and (c) the second bolus of GdDOTP5⫺. Three ROIs within the kidney are considered: the cortex (solid), medulla
(dotted), and calyx-ureter (dashed). Signal enhancement following the first GdDOTP5⫺ bolus was converted to concentration by using r1 ⫽
3.0 mM⫺1 s⫺1. Residual enhancement from the previous bolus(es) of contrast agent was estimated by extrapolation and subtracted out
prior to calculation of the relaxation rate enhancement in panel b and the AUC/time concentration of GdDOTP5⫺ in panel c.
demonstrate that it is possible to reproduce the pharmacokinetics of GdDOTP5⫺ in a given animal.
For the animal in Fig. 4, Fig. 5 shows an AUC/time
apparent concentration map of GdDOTP5⫺ at 30 min
postinjection following the first bolus (Fig. 5a), the final
bolus (Fig. 5b), and the absolute difference image of the
two (Fig. 5c). Excellent agreement between the two boluses
can be seen for pixels falling within the kidneys, except for
a small region near the proximal end of the ureter draining
the right kidney, and also at the edges of the kidneys.
Agreement between the two images is somewhat poorer in
the extrarenal pixels, possibly due to poor signal enhancement in these regions at the dosage of contrast agent used.
In Vivo pH Calculations
We observed that the renal pharmacokinetics of GdDTPA2⫺ and GdDOTP5⫺ are similar (Fig. 3 and Table 1).
We also observed excellent agreement between the
wash-in kinetics of GdDOTP5⫺ and GdDOTA-4AmP5⫺ in
the renal cortex, medulla, and calyx-ureter (Fig. 4 and
Table 2
The Pharmacokinetics of GdDOTP5⫺ and GdDOTA-4AmP5⫺,
and the Reproducibility of the Pharmacokinetics of GdDOTP5⫺,
in SCID Mice
ROI
Mouse 1
Cortex
Medulla
Ureter
Mouse 2
Cortex
Medulla
Ureter
Mouse 3
Cortex
Medulla
Ureter
t90 (min)
GdDOTP5⫺
t90 (min)
GdDOTA4AmP5⫺
t90 (min)
GdDOTP5⫺
SD/
mean
7.3
9.0
9.3
7.6
9.7
10.6
8.6
9.3
10.0
0.071
0.031
0.053
8.7
9.3
9.6
8.8
10.6
11.2
8.4
9.8
10.2
0.020
0.054
0.064
8.4
9.7
10.7
7.2
8.6
10.1
7.8
8.9
9.4
0.063
0.051
0.053
Table 2). Based on these observations, and also on the
similarity of molecular structure and charge between GdDOTP5⫺ and GdDOTA-4AmP5⫺ (Fig. 1), we made the assumption that the MR pharmacokinetics of GdDOTP5⫺ can
be used as a surrogate for the pharmacokinetics of GdDOTA-4AmP5⫺ in a given animal. Tissue pH maps were
calculated from the AUC/time at 30 min postinjection of
GdDOTP5⫺ and GdDOTA-4AmP5⫺ in a given mouse, and
the pH-dependent relaxivity of GdDOTA-4AmP5⫺ measured in vitro. The pharmacokinetics of the two boluses of
GdDOTP5⫺ were averaged prior to calculation of pH. Figure 6b shows a representative pH image of the kidneys and
nearby tissues in a control mouse. A fat-suppressed proton-density-weighted image is shown in Fig. 6a for anatomic referencing. The bulk of the pixels falling within the
kidneys have a calculated pH of 7.0 –7.4, whereas the
pixels corresponding to the papilla and calyx are acidic
(pH 5.75–7.0). Most of the extrarenal pixels appear to be at
near-neutral pH, although acidic pixels can also be seen in
these regions. It should be noted that in our calculations
tissue regions below ⬇pH 5.75 would be misassigned to a
higher pH on the other side of the inflection in the titration
in Fig. 2. From Table 1 we see that the variability between
FIG. 5. Images depicting the AUC/time apparent concentration
maps of GdDOTP5⫺ at 30 min postinjection for the animal in Fig. 4:
(a) first bolus, and (b) last bolus. The absolute difference image 兩a ⫺
b兩 is displayed in panel c at 100% higher intensity. Pixels that were
not ⱖ 5% of the signal intensity of the brightest pixel in the precontrast image, and that did not undergo at least an average of 50%
enhancement in signal 5–10 min following the first bolus, were not
processed and were set equal to background.
256
Raghunand et al.
FIG. 6. pH image of a control female SCID mouse: (a) fat-suppressed, proton-density-weighted reference anatomic image showing a coronal view of the kidneys and nearby tissues; (b) corresponding calculated pH image. A pH of 6.0 was measured in a urine
sample collected immediately following the imaging experiment.
successive boluses in a given animal is of the order of 10%
in the cortex and medulla, and higher in the calyx-ureter.
After averaging the pharmacokinetics of the two GdDOTP5⫺ boluses, this would translate into an estimated
variation of ⫺0.25 to ⫹0.20 in the calculated pH at a true
pH of 7.2, for example. In three control mice, we measured
a mean renal cortical pH of 7.3 ⫾ 0.13, a mean medullary
pH of 7.0 ⫾ 0.29, a mean pH in the calyx-ureter ROI of
6.3 ⫾ 0.45, and a mean pH of urine collected immediately
following the MR examination of 5.9 ⫾ 0.10 (Table 3).
Acetazolamide, a carbonic anhydrase inhibitor, causes
systemic metabolic acidosis and alkalinization of urine
because the kidneys of treated mice are unable to reclaim
all the filtered bicarbonate. Figure 7 shows a pH image of
the kidneys and nearby tissues in an acetazolamide-treated
mouse. Consistent with its expected action, acetazolamide
appears to raise the pH of renal pixels by 0.4 –1.1 pH units
in this animal compared to the control mouse in Fig.
6. The pH values of nonrenal tissues also appear to be
more acidic than equivalent tissues in the control mouse.
It must be kept in mind that the relatively low signal
enhancement in nonrenal pixels means that the uncertainty in the calculated pH values in nonrenal pixels is
higher than in renal pixels. Some areas of seemingly very
low pH are also seen in areas of Fig. 7 with low precontrast
signal intensity, such as those along the expected locations
of major blood vessels or near “fatty” areas that were
suppressed by fat presaturation. These artifacts likely arise
FIG. 7. pH image of an acetazolamide-treated mouse. a: T1weighted, postcontrast reference anatomic image showing the kidneys and nearby tissues. The cortex, medulla, calyx, and proximal
segments of both ureters are visible. b: Corresponding calculated
pH image. A pH of 8.2 was measured in a urine sample collected
immediately following the imaging experiment.
from partial-volume effects. In two acetazolamide-treated
mice the mean renal cortical pH was 7.7 ⫾ 0.18, the mean
medullary pH was 8.0 ⫾ 0.13, the mean pH in the calyxureter ROI was 7.5 ⫾ 0.21, and the mean pH of urine
collected immediately following the MR examination was
8.0 ⫾ 0.19 (Table 3). The increases in medullary and urine
pH vis-à-vis control mice were statistically significant despite the small sample sizes.
CONCLUSIONS
Gene therapies show promise in the treatment of inherited
and acquired renal tubular acidosis (8,9), and methods to
improve the delivery and retention of corrective genes to
the kidneys are being developed in animal models of the
disease (29). In the near term, pH imaging applied to
animal models of renal tubular acidosis will permit the
noninvasive assessment of renal function, provide spatial
information on the uniformity of corrective gene delivery,
permit longitudinal assays of gene retention and activity
on the same cohort of mice, and permit comparison of two
different therapies applied to contralateral kidneys in the
same animal. Potential clinical applications for pH imaging by MRI can also be envisioned for the longer term.
Microenvironmental pH also impacts tumor response to
chemotherapy with weakly-ionizing drugs (5), and knowledge of tumor pH distribution is important, particularly
with cancers of tissues with normally acidic regions, such
Table 3
Renal pH in Control and Acetazolamide-Treated SCID Mice
Control
Mouse 1
Mouse 2
Mouse 3
Mean ⫾ SD
Acetazolamide-treated
Mouse 1
Mouse 2
Mean ⫾ SD
P vs. Control
a
pH, cortexa
pH, medullaa
pH, calyx-uretera
pH, urine
7.11
7.27
7.44
7.3 ⫾ 0.13
6.62
7.22
7.23
7.0 ⫾ 0.29
6.88
6.34
5.78
6.3 ⫾ 0.45
5.88
6.03
5.79
5.9 ⫾ 0.10
7.47
7.82
7.7 ⫾ 0.18
0.13
7.86
8.13
8.0 ⫾ 0.13
0.04
7.25
7.66
7.5 ⫾ 0.21
0.2
7.83
8.21
8.0 ⫾ 0.19
0.001
Average pH in hand-drawn ROIs, in both kidneys and imaging slices.
pH Imaging Using GdDOTA-4AmP5⫺
as renal cell carcinoma and bladder carcinoma (30). This
study demonstrates the feasibility of imaging tissue pH in
mice by contrast-enhanced MRI using the pH-sensitive
contrast agent GdDOTA-4AmP5⫺. The pH images of acetazolamide-treated mice were consistent with the expected
activity of the drug on renal tubular ion transport. Studies
are underway to further characterize and minimize
sources of error in the dual-contrast-agent approach reported here.
ACKNOWLEDGMENTS
This work was partially funded by grants from the American Cancer Society (IRG7400124-451224 to N.R.) and the
NIH (R21-CA84697 to A.D.S., and R01-CA77575 and R24CA83148 to R.J.G.).
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