Gadolinium Et Fibrose

Rôle des complexes de gadolinium dans le mécanisme de
la fibrose systémique néphrogénique

Nathalie Fretellier
To cite this version:

Nathalie Fretellier. Rôle des complexes de gadolinium dans le mécanisme de la fibrose systémique
néphrogénique. Médecine humaine et pathologie. Université René Descartes - Paris V, 2013. Français.
�NNT : 2013PA05P609�. �tel-00843108�
HAL Id: tel-00843108

https://theses.hal.science/tel-00843108
Submitted on 10 Jul 2013
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ORIGINAL ARTICLE
Comparative In Vivo Dissociation of Gadolinium Chelates in

Renally Impaired Rats
A Relaxometry Study
Nathalie Fretellier, MS,* Jean-Marc Idée, PharmD,* Anne Dencausse, PharmD,* Oussama Karroum, PhD,*
Sylviane Guerret, PhD,† Nicolas Poveda, MS,* Gaëlle Jestin, BS,* Cécile Factor, PhD,*
Isabelle Raynal, PhD,* Philippe Zamia, MS,* Marc Port, PhD,* and Claire Corot, PharmD*
Conclusions: Unlike Dotarem, Omniscan and gadodiamide induced histo-

Purpose: Investigation of dissociated versus chelated gadolinium (Gd) in logic skin lesions. At day 11, a higher Gd concentration was found in both
plasma, skin, and bone of rats with impaired renal function after adminis- skin and femur of Omniscan- and gadodiamide-treated rats than in Dotarem-
tration of ionic macrocyclic (gadoterate or Dotarem) or nonionic linear treated rats. Relaxometry results indicate gradual in vivo dechelation and
(gadodiamide or Omniscan) Gd chelates. release of dissociated Gd31 in a soluble form in renally impaired rats
Materials and Methods: Subtotally nephrectomized Wistar rats were sub- receiving Omniscan and gadodiamide, whereas Dotarem remained stable
jected to receive daily injections of 2.5 mmol/kg of Omniscan, gadodiamide over the study period.
without excess ligand caldiamide, Dotarem, or saline (n 5 7–10 rats/group)
for 5 consecutive days. The Gd concentration was measured by inductively Key Words: nephrogenic systemic fibrosis, gadolinium chelates,
coupled plasma mass spectrometer in skin, femur epiphysis, and plasma on gadodiamide, gadoterate, renal impairment, relaxometry
completion of the study (day 11), and dissociated Gd31 was measured in the (Invest Radiol 2011;46: 292–300)
plasma at day 11 (liquid chromatography inductively coupled plasma mass
spectrometry). The r1 relaxivity constant was measured in skin (at day 4 and
day 11) and bone (day 11) to investigate the dissociated or chelated form of
Gd found in tissue samples. Clinical and skin histopathologic studies were
performed.
Results: Subtotal nephrectomy decreased creatinine clearance by 60%. No
M any studies have demonstrated the presence of gadolinium
(Gd) in skin biopsies from patients who experienced nephro-
genic systemic fibrosis (NSF) after administration of a Gd chelate
macroscopic skin lesions were observed in the Dotarem and Omniscan (GC), by energy dispersive x-ray spectroscopy combined with scan-
groups in contrast with the gadodiamide group (2 rats survived the study ning electron microscopy,1– 4 energy-filtered transmission electron
period and 4 of 10 rats showed skin ulcerations and scabs). Skin histopatho- microscopy with electron energy loss spectroscopy,5,6 synchrotron
logic lesions were in the range gadodiamide . Omniscan . Dotarem x-ray fluorescence spectroscopy,7 or mass spectrometry.8,9 Unfor-
(similar to control rats). At day 11, the skin Gd concentration was lower in tunately, although it is very unlikely that a water-soluble GC would
the Dotarem group (161.0 6 85.5 nmol/g) as compared with the Omniscan persist for a very long time in skin samples, especially after processing
(490.5 6 223.2 nmol/g) and gadodiamide groups (mean value, 776.1 nmol/g; with solvents,10 the techniques used in these studies are unable to
n 5 2 survivors). The total Gd concentration in the femur was significantly formally discriminate between chelated and dissociated Gd.
higher in the Omniscan group than in the Dotarem group. At day 11, the The mechanism of NSF would be considerably elucidated by
dissociated Gd31 concentration in plasma was below the limit of detection in determining the dissociated versus chelated, and soluble versus
the Dotarem group and was 1.5 6 0.7 mmol/L in the Omniscan group insoluble states of Gd found in biopsy samples. For example, in
corresponding to 62% 6 15% of the total Gd concentration. The dissociated patients with renal insufficiency and pro-inflammatory states, it has
Gd31 concentration was 1.1 mmol/L in gadodiamide rats (n 5 2 survivors). been proposed that free Gd may precipitate with phosphate to form
In the skin, the in vivo r1 relaxivity value increased from 4.8 6 0.7 mM21s21 insoluble salts that may be phagocytosed by macrophages which
at day 4 to 10.5 6 3.9 mM21s21 at day 11 in the Omniscan group, P , 0.05 could subsequently release chemokines and cytokines involved in
(in vitro r1 in skin, 3.5 mM21s21) and gadodiamide group, whereas no the attraction and activation of circulating fibrocytes.6,11–13 Alter-
significant change was observed in the Dotarem group (2.8 6 0.2 and 4.9 6 natively, other authors have reported a direct stimulating effect of
2.8 mM21s21 at day 4 and 11, respectively, NS) (in vitro value in the skin, “free,” soluble Gd31 on fibroblast proliferation in vitro.14,15 It has
3.2 mM21s21). In the femur, the in vivo r1 relaxivity was higher in the also been speculated that dissociated and soluble Gd31 may interfere
Omniscan group (8.9 6 2.1 mM21s21) (in vitro relaxivity, 4.5 mM21s21) with fibrillogenesis and hence with collagen production in the
and gadodiamide group (8.8 mM21s21, n 5 2 survivors) than in the Dotarem dermis.16 On the other hand, on the basis of in vitro studies
group (3.8 mM21s21, n 5 1 rat with measurable r1, since for 7 rats, 1/T1 2 performed at high GC concentration,17 Newton and Jiménez have
1/T1(diamagnetic) ,10% of 1/T1(diamagnetic) because of low Gd concentration) speculated that the GC molecules themselves (ie, without in vivo
(in vitro relaxivity value in the femur matrix, 3.1 mM21s21). dissociation) may have a direct pro-inflammatory and profibrotic
effect.18
The aim of this study was to characterize the dissociated or
chelated state of Gd in plasma, skin, and trabecular bone after
Received July 28, 2010; accepted for publication (after revision) September 30,
2010.
administration of marketed GC, representative of 2 distinct catego-
From the *Research Division, Guerbet, Roissy Charles de Gaulle Cedex, France; ries with opposite physicochemical properties: a linear, nonionic GC
and †Pathology Department, Novotec, Lyon, France. (both low thermodynamic and kinetic stabilities) and an ionic,
Reprints: Nathalie Fretellier, MS, Research Division, Guerbet, BP 57400, 95943 macrocyclic GC (both high thermodynamic and kinetic stabili-
Roissy Charles de Gaulle Cedex, France. E-mail: nathalie.fretellier@
guerbet-group.com.
ties)19,20 to rats with impaired renal function. Plasma total Gd concen-
Copyright © 2011 by Lippincott Williams & Wilkins tration was measured by inductively coupled plasma mass spectrometry
ISSN: 0020-9996/11/4605-0292 (ICP-MS), and dissociated Gd concentration was measured by high
292 | www.investigativeradiology.com Investigative Radiology • Volume 46, Number 5, May 2011

Investigative Radiology • Volume 46, Number 5, May 2011 Dissociation of GC in Renally Impaired Rats
performance liquid chromatography (HPLC) coupled to an ICP-MS potential lesions. The backs and abdomens were shaved again at
system.21 Because HPLC-ICP-MS cannot be used in solid biologic completion of the study. Skin samples were taken from the backs of
matrices such as skin or bone tissues for practical reasons, another animals (normal areas and areas presenting lesions).
approach, relaxometry, was selected. For that purpose, we assumed A terminal blood sample was obtained, and skin of the back
that in vivo relaxometry of tissue samples (skin and bone) would be and bone (femur epiphysis) were harvested for Gd assay and
able to discriminate between the dissociated, chelated, or insoluble relaxometry studies.
state of Gd, based on their different effects on the relaxation times All injections, blood, and skin sampling were performed
of adjacent proton spins.22,23 under isoflurane/oxygen (3.5% induction, then 2.5%, 1 L/min oxy-
gen) anesthesia.
MATERIALS AND METHODS Histology

Histopathologic examinations were performed under blinded
Animal Study conditions by a certified histopathologist (S.G.) in accordance with
All studies were carried out on male, Wistar rats from CERJ the guidelines of the Society of Toxicologic Pathology.26
(Centre d’Elevage René Janvier, Le Genest Saint Isle, France), aged Skin samples were fixed in 4% neutral buffered formalin.
6 weeks and weighing 235 6 23 g. After routine dehydration, the samples were embedded in paraffin,
The animals underwent subtotal (5/6th) nephrectomy sectioned (5 mm thickness) for hematoxylin-eosin-saffron staining
(SNx)24,25: the right kidney was exposed through a flank incision, for topographic analysis, picrosirius red staining to detect the pres-
the adrenal gland was separated from the upper pole, and the kidney ence of collagen, and Alcian blue staining to detect acidic mucopo-
was decapsulated. The renal pedicle was ligated and the right kidney lysaccharides. Immunohistochemical staining was performed to de-
was removed. The left kidney was subsequently decapsulated, and tect CD34 and transforming growth factor (TGF)-b1 positive cells
the adrenal gland was separated from the upper pole. Ligatures were using anti-CD34 goat antibody (diluted 1:1000, R&D Systems,
placed around the upper and lower poles and the poles were excised. Minneapolis, MN) and anti-TGF-b1 rabbit antibody (diluted 1:250,
Animals were anesthetized with ketamine and xylazine. An intrave- GeneTex, San Antonio, TX) using the ImmPRESS polymerized re-
nous injection of 1.0 mL/kg of saline was administered at comple- porter peroxidase staining (ImmPRESS antigoat Ig @peroxidase# kit
tion of surgery to compensate for blood loss. The surgical procedure @MP 7405# and ImmPRESS antirabbit Ig @peroxidase# kits @MP 7401#,
was carried out at the CERJ laboratory. respectively (Vector Laboratories, Burlington, CA).
The animals were housed 1 per cage at an ambient tempera-
ture of 22°C 6 2°C, hygrometry of 55% 6 10%, with 12/12 Gadolinium Measurement
light/dark cycles. Animals had free access to water and standard Gd was measured in plasma, skin, and trabecular bone sam-
animal chow (reference A04, SAFE, Augy, France) containing (per ples byICP-MS on ELAN DRC Plus (PerkinElmer Life and Ana-
kilogram) 5.9 g phosphorus, 8.3 g calcium, 2.5 g sodium, 6.7 g lytical Sciences, Inc., Waltham, MA). A standard curve of inorganic
potassium, 2 g magnesium, 85 mg zinc, and 240 mg iron. The Gd (0.64 nmol/L–1.30 mmol/L) in 6.5% HNO3 was used by mon-
protein concentration was 16.1%. itoring the response of the 157Gd isotope. The acceptance limits
All experimental procedures were carried out according to the (total error) were set at 614%. Results are expressed in nmol of Gd
French rules on animal welfare and complied with the European per gram tissue wet weight (skin and bone samples) or mmol per liter
Economic Community Directive (2010/63/EU). The protocol was of plasma. The limit of quantification (LOQ) was 0.64 nmol/L.
approved by the local ethics committee. Samples above the limit of detection (LOD) (0.19 nmol/L) but
A total of 33 male, SNx Wistar rats were allocated to daily below the LOQ were given an arbitrary value of 0.32 nmol/L.
treatment with either saline (5.0 mL/kg), 500 mM Dotarem (meglu- The plasma dissociated Gd31 concentration was measured by
mine gadoterate; Guerbet, Villepinte, France, batch 8GD051B), 500 HPLC connected to an ICP-MS system according to Frenzel et al.21
mM Omniscan (gadodiamide; GE Healthcare, Chalfont St Giles, The HPLC system (Series 200 LC PerkinElmer Life and Analytical
United Kingdom, batch 10758384), or nonformulated Omniscan (ie, Sciences, Inc., Waltham, MA) was equipped with a 1-mL chelating
without the 25 mM excess ligand caldiamide present in the com- Sepharose column (HiTrap, GE Healthcare, Uppsala, Sweden) and
mercial solution of Omniscan) (495 mmol/L, synthesized at the was connected to an ICP-MS system (ELAN DRC Plus). Five-mL
Guerbet Research Department). We shall use the term “Omniscan” aliquots (a 300-mL sample was needed to take the dead volume into
for the marketed product, and the term “gadodiamide” for the pure account) were injected into the column which was equilibrated with
drug substance. We shall use the term “Dotarem” as a synonym of 10 mmol/L bis-Tris buffer (pH 6). The HPLC flow rate was set at 1
meglumine gadoterate. The rats received saline or 2.5 mmol/kg of mL/min.
each GC intravenously through the tail vein at a rate of 1.0 mL/min, The signal intensity of the 157Gd isotope was continuously
daily for 5 consecutive days, starting 10 days after subtotal nephrec- recorded and displayed on a chromatogram. The fraction of free
tomy. This period was defined during a preliminary study in which Gd31 was measured by peak area analysis.
10 SNx rats were compared with 4 control, sham-operated animals. The linear response curve and recovery of the released free
The creatinine clearance of the SNx rats was found to be signifi- Gd31 were measured by testing increasing concentrations of Gd
cantly decreased from the first measurement (day 7) when compared nitrate (1, 5, 10, 20, 50, and 100 mM) in water and 0.5, 1, 2, 5, and
with sham-operated rats (P , 0.05). Renal function remained stable 10 mM of Gd nitrate in rat plasma.
in the SNx group throughout the study (0.38 – 0.48 mL/min/100 g The stability over time was checked during the sequence by
body weight). Creatinine clearance was 1.0 to 1.2 mL/min/100 g quality controls (theoretical concentration 5 4 mmol/L, measured
body weight in the sham-operated control group. mean value, 3.71 6 0.08 mmol/L @n 5 5#) (mean 6 standard
The rats were killed (exsanguination under isoflurane anes- deviation @SD#). The LOQ of the method was 0.5 mmol/L.
thesia) 11 days after the first administration.
The rats were examined for macroscopic skin changes before Relaxometry Measurements
the first injection and then daily throughout the study. On day 4, the Both epiphyses from 1 femur (about 500 mg) and a dorsal
backs of the animals were shaved for biopsy (6-mm diameter biopsy skin sample (biopsy, 30 mg and at sacrifice, 500 mg) were used for
punch, Labonord, Templemars, France) and better visualization of relaxometry measurements. One 1:11 dilution was performed in
© 2011 Lippincott Williams & Wilkins www.investigativeradiology.com | 293

Fretellier et al Investigative Radiology • Volume 46, Number 5, May 2011
milliQ water to mash samples. Mashing of samples differed accord- Relaxometry studies (spiking in vitro studies) on biologic
ing to the matrix: bone was first placed in the liquid nitrogen matrices were performed by spiking with Dotarem and Omniscan
(2196°C) and subsequently crushed manually. Large amounts of (both from commercial solutions, 500 mM) (range of 0, 0.05, 0.1,
matrices (bone and skin) were first mashed with an Ultra-Turrax 0.25, 0.5, and 1 mM) on water, rat plasma, skin, and femur epiphysis
homogenizer (IMLAB, Lille, France) followed by a Precellys homog- from nontreated rats (final test volume, 300 mL) at a proton fre-
enizer (Bertin technologies, Montigny-le-Bretonneux, France). Small quency of 60 MHz and 37°C. Gd acetate (408 mM, similar concen-
amounts of matrices were mashed with a Precellys homogenizer. tration range as GC) was used to investigate the effect of free,
Before measurement, the suspension was mixed to avoid sedimentation soluble Gd. The effect of precipitated, amorphous Gd was tested
and was eventually checked for a homogeneous aspect. with phosphate, hydroxide, and carbonate salts of Gd (5 mg in 100
Relaxometry study was performed (100 mL sample) with a mL of MilliQ water or 100 mL of skin matrix sample). Relaxometry
Minispec mq 60 (Bruker Optics, Ettlingen, Germany), operating at studies were performed immediately after spiking the matrix. Gd
a proton frequency of 60 MHz, and a preset temperature of 37°C 6 concentration was measured by ICP-MS.
2°C). The Minispec standard software was a 2-pulse inversion- We termed all experiments performed by spiking GC on
recuperation spin echo with a fixed relaxation delay of 5 3 T1. tissue matrices (to obtain the reference range of r1 relaxivities) as “in
When the 1/T1 2 1/T1(diamagnetic) value was less than 10% vitro studies” and all the experiments that were performed on test
of 1/T1(diamagnetic) in the absence of Gd precipitation (ie, because animals as “in vivo studies.”
of a low total Gd concentration in the sample), the r1 relaxivity could On the basis of in-house in vitro relaxometry studies (Dota-
not be measured. rem in water, 37°C, at 20 MHz), the uncertainty of relaxivity r1
The influence of mashing of skin and bone tissues on r1 measurements (Gd concentration measured by ICP-MS) was set at
relaxivity was studied to ensure that there was no alteration in the r1 6 23%. The in vitro r1 relaxivity range represents the in vitro r1
molecular form of the GC present in the sample. The r1 relaxivity relaxivity value (obtained from spiking studies) 6 uncertainty of 23%.
was measured before and after mashing of samples with the Pre- For ICP-MS and relaxometry studies, tissue samples were
cellys and/or the Ultra-Turrax homogenizer. Dotarem remained stored at 280°C.
stable regardless of the mashing technique. Omniscan remained
stable after Ultra-Turrax mashing but was found to be degraded Statistical Analysis
(overlapping ratio of 124%, ie, an increase in the r1 value from 3.8 Data are expressed as mean 6 SD. SD was given only for
to 4.7 mM21s21) after 8 mashing sequences with the Precellys samples .2 rats. The in vivo relaxivity r1 values were compared
homogenizer. The mashing technique was therefore modified (2 using a t test. The 95% confidence intervals on the in vivo relaxivity
sequences at 15-minute interval) to avoid this degradation. r1 mean were calculated (mean 6 2 standard error of the mean) and
The GC concentration in tissue samples was corrected for were compared with the in vitro relaxivity r1 values 6 23% (in vitro
water content with a proportion of 97% water being estimated on the r1 range). If the confidence interval on the in vivo relaxivity r1 mean
basis of unpublished studies. All samples were measured individu- was found to be fully included in the in vitro relaxivity r1 range, the
ally at 1.42 T on 100 mL samples. in vivo Gd state was considered equivalent to the in vitro Gd state
(chelated, dissociated, or precipitated according to the respective
relaxivity r1 value), and otherwise different.
Assuming a normal distribution of values, Gd concentration
TABLE 1. Macroscopic Skin Lesions and Other Clinical measurements were tested globally with analysis of variance with
Symptoms Observed After Treatment With Dotarem, repeated measures. In the case of a significant difference, pairwise
Omniscan, Gadodiamide, or Saline (5 3 2.5 mmol/kg Daily) comparisons were performed using a t test.
No. Live Rats Statistical analyses were carried out using GraphPad Prism
No. Rats at Completion No. Rats With software (GraphPad Software Inc, San Diego, CA) or SAS (Statis-
per of the Study Macroscopic Other tical Analysis Software, Cary, NC). A P # 0.05 was considered to
Treatment Group (Day 11) Skin Lesions Symptoms indicate a significant difference.
Saline 7 7 0 —
Dotarem 8 8 0 — RESULTS
Omniscan 8 8 0 —
Clinical and Histologic Data
Gadodiamide 10 2 4 (scab Prostration,
formation piloerection,
Clinical data are shown in Table 1. Epidermal lesions (ulcer-
and loss of ations and scabs) on the neck, back, and abdominal skin were found
ulceration) bodyweight in 4 of the 10 rats treated with gadodiamide. These lesions were
observed for the first time at day 5 (ie, the day of the last injection)
TABLE 2. Summary of Skin Histologic Findings

Treatment Summary of Histologic Findings
Control Very few histopathologic lesions. Dermis: constant thickness and low cellularity. Extracellular matrix: dense underneath the epidermis
and homogeneous in the dermis. Some CD341 or TGFb11 positive cells.
Dotarem Very few histopathologic lesions. Dermis: constant thickness and low cellularity. Extracellular matrix: homogeneous. Some CD341
or TGFb11 positive cells.
Omniscan Small superficial epidermal lesions and degradation of collagen fibers in the dermis with disorganization of the extracellular matrix.
Some CD341 or TGFb11 positive cells.
Gadodiamide Numerous superficial epidermal lesions, inflammation, necrosis, and increased cellularity, as well as large areas of complete degradation
of the dermal extracellular matrix. CD34 or TGFb1 positive staining on spindle-shaped cells and dendrocytes in the dermis.
294 | www.investigativeradiology.com © 2011 Lippincott Williams & Wilkins

for 2 rats and at day 9 for 2 rats. In the gadodiamide group, 8 rats CD341 or TGFb11 positive cells after immunostaining were found
were either found dead or had to be killed for ethical reasons. The on spindle-shaped cells and dendrocytes in the dermis of all test
2 surviving rats treated with gadodiamide experienced a loss of groups, including the control rats (Table 2).
body weight.
No macroscopic skin lesions were found in the rats treated Total and Dissociated Gadolinium Levels in the
with Dotarem or Omniscan or in the control group. No significant Plasma
difference in body weight changes was observed between these Plasma total Gd concentrations at days 1, 5, and 11 are shown
groups. Briefly, very few histologic lesions were reported in the in Table 3.
Dotarem-treated group or in the control group. Small superficial Dissociated Gd concentrations (measured by HPLC-ICP-MS)
epidermal lesions and some degradation of collagen fibers in the are shown in Figure 1. The dissociated Gd concentration measured
dermis were observed in the Omniscan group. Numerous superficial in the plasma of Dotarem-treated rats at the time of sacrifice was
epidermal lesions, inflammation, necrosis, and increased cellularity below the limit of detection (LOD) value but was 1.4 6 0.7 mmol/L
as well as large areas of complete degradation of the dermal in Omniscan-treated rats and 1.1 mmol/L (n 5 2 rats) in the
extracellular matrix were observed in the gadodiamide group. gadodiamide group.
The dissociated/total Gd concentration ratio was 62.0% 6
15.0% in the Omniscan group, and 52.9% in the gadodiamide group.
TABLE 3. Total Gadolinium Concentration in Rat Plasma at No dissociated Gd was detected in the Dotarem group (all values ,
Days 1, 5, and 11 (ICP-MS) (Mean 6 SD) LOD).
Gadolinium Concentration (No. Rats) Total Gadolinium Levels in Skin
Day 1 Day 5 Day 11 When measured at day 4 (ie, during the injection period), total
(mmol/L) (mmol/L) (mmol/L) Gd concentration in skin samples was statistically similar in the
Dotarem, Omniscan, and gadodiamide groups. At day 11, a dramatic
Control 1026 6 2.1026 0.77 6 0.99 0.01 6 0.02
decrease in the Gd concentration was measured in all treated groups
(n 5 7) (n 5 7) (n 5 7) (Fig. 2). The Gd concentration was lower in the Dotarem group
Dotarem 11.1 6 1.2 6.8 6 8.4 3.3 6 3.1 (161.0 6 85.5 nmol/g) than in the Omniscan (490.5 6 223.2
(n 5 8) (n 5 8) (n 5 8) nmol/g) and the gadodiamide groups (mean, 776.1 nmol/g, n 5 2),
Omniscan 11.1 6 1.5 5.6 6 2.6 2.3 6 1.4 but this difference was not statistically significant.
(n 5 8) (n 5 8) (n 5 8)
Gadodiamide 11.9 6 1.9 12.7 6 10.4 2.0 Total Gadolinium Levels in Femur
(n 5 10) (n 5 8) (n 5 2) The total Gd concentration was higher in the femur of the 2
surviving rats treated with gadodiamide (mean, 631.4 nmol/g) than
ICP-MS indicates inductively coupled plasma mass spectrometry; SD, standard in the animals of the Dotarem (98.8 6 53.4 nmol/g) and Omniscan
deviation.
groups (353.1 6 81.4 nmol/g). The total Gd concentration in the
FIGURE 1. Dissociated Gd concentration in

plasma at sacrifice (day 11) (HPLC-ICP-MS
measurement, n 5 8 rats/group except n 5 2
for gadodiamide and 7 for saline). LOD indi-
cates limit of detection.
FIGURE 2. Total Gd concentration

observed in skin samples of subto-
tally (5/6th) nephrectomized rats at
day 4 (biopsy) and day 11 (sacri-
fice) (ICP-MS measurement). The
rats received intravenous injections
of 2.5 mmol/kg/d of GC or 5 mL/
kg/d of saline for 5 consecutive
days.

FIGURE 3. Total Gd concentration in bone

(femur epiphysis) tissue of rats with subtotal
(5/6th) nephrectomy at day 11 (ICP-MS mea-
surement, n 5 8 rats/group except n 5 2 sur-
viving rats for gadodiamide and 7 rats for sa-
line). The rats received intravenous injections
of 2.5 mmol/kg/d of GC or 5 mL/kg/d of sa-
line for 5 consecutive days. *P , 0.05.
TABLE 4. r1 Relaxivity Value (Spiking Studies With TABLE 5. r1 Relaxivity Value (60 MHz, 37°C) of 3
Dotarem, Omniscan, and Gd Acetate (60 MHz, 37°C) in Precipitated Gd Salts in Water and Skin
Water and Various Tissue Matrices
r1 Relaxivity (mM21s21) in
Ex Vivo r1 Relaxivity (mM21s21)
Water Skin
Matrix Dotarem Omniscan Gd acetate Gd phosphate 0.04 (0.03–0.05) 0.02 (0.015–0.025)
Water 3.0 (2.3–3.7) 3.1 (2.4–3.8) 10.4 (7.6–12.8) Gd hydroxide 0.29 (0.22–0.36) 0.29 (0.22–0.36)
Plasma 3.7 (2.9–4.6) 4.1 (3.2–5.0) 15.4 (11.9–19.0) Gd carbonate 0.01 0.08 (0.06–0.10)
Trabecular femur 3.1 (2.4–3.8) 4.5 (3.5–5.5) 15.8 (12.2–19.4)
Gd indicates gadolinium.
Skin 3.2 (2.5–4.0) 3.5 (2.7–4.3) —
Gd indicates gadolinium.
values measured in femur samples of both Omniscan and gadodi-
amide were higher than those in the vitro range.
femur was higher in the Omniscan group than in the Dotarem group Relaxometry Values in the Plasma
(P , 0.05) (Fig. 3). As the 1/T1 2 1/T1(diamagnetic) value was less than 10% of
Relaxometry Studies the 1/T1 (diamagnetic) value for all plasma samples at day 11, no in
The in vitro r1 relaxivity values obtained in water, rat plasma, vivo r1 relaxivity value was obtained in the plasma, regardless of
skin, and trabecular femur matrices (spiking studies with Dotarem, treatment.
Omniscan, and Gd acetate) are shown in Table 4.
The in vitro r1 relaxivity values of precipitated forms of Gd DISCUSSION
(phosphate, hydroxide, and carbonate) in water and in skin are Subtotal nephrectomy in rats is classically described as a
shown in Table 5. model of chronic kidney disease and renal fibrosis,24,25 and this
model has been widely used to investigate the profibrotic effects of
Relaxometry Values in the Skin GC.27–29 As described in these studies, a repeated GC injection
On in vivo studies, the skin r1 relaxivity value increased from schedule was used in the present study. At 2.5 mmol/kg, the dose of
4.8 6 0.7 mM21s21 at day 4 to 10.5 6 3.9 mM21s21 at day 11 in GC used in the rat was higher, per unit body weight, than the dose
the Omniscan group (P , 0.05 vs. day 4), whereas no significant range of 0.1 to 0.3 mmol/kg classically used for magnetic resonance
change was observed in the Dotarem group (2.8 6 0.2 and 4.9 6 2.8 imaging contrast procedures in patients.30 However, this dose range
mM21s21 at days 4 and 11, respectively, NS) (Fig. 4). In the was considered to be appropriate for chronic studies in rats, as
gadodiamide group, the r1 value was 5.5 6 1.2 mM21s21 at day 4 comparative drug doses between species must be normalized to
(n 5 8) and 11.4 mM21s21 at sacrifice (mean for n 5 2 survivors). body surface area rather than body weight. The US Food and Drug
At day 11, the r1 relaxivity values measured in skin samples of the Administration recommends the use of a 6-fold increase to obtain a
Omniscan and gadodiamide groups were higher as compared with human equivalent drug dose for rats.31 On this basis, a dose of 0.4
those in vitro range. The r1 relaxivity value for Dotarem was mmol/kg GC in human being would be equivalent to 2.5 mmol/kg in
included in the in vitro range. the rat.
Two structurally distinct categories of GC are currently mar-
Relaxometry Values in the Femur keted: (a) “macrocyclic” chelates (gadoterate or Dotarem, gadoteri-
In the trabecular femur, the in vivo r1 relaxivity value was dol or ProHance, gadobutrol or Gadovist), in which the Gd31 ion is
higher in the Omniscan group (8.9 6 2.1 mM21s21) (in vitro value: “caged” in the preorganized cavity of the ligand, and (b) “linear”
4.5 mM21s21) and gadodiamide group (8.8 mM21s21) than in the chelates (gadopentetate or Magnevist, gadobenate or MultiHance,
Dotarem group (3.8 mM21s21) (Fig. 5). In the Dotarem group, only gadodiamide or Omniscan, gadoversetamide or OptiMARK, gado-
1 rat had measurable r1, as the 1/T1 2 1/T1(diamagnetic) value was fosveset or Ablavar, and gadoxetate or Eovist/Primovist). GC can
less than 10% of 1/T1(diamagnetic) in the remaining 7 rats due to the also be either “nonionic” (in which the number of carboxyl groups
low total Gd concentration (8 6 2 mmol/L). The in vivo r1 relaxivity is reduced to 3, neutralizing the 3 positive charges of the Gd31) or

FIGURE 4. Relaxivity in vivo r1 apparent (app.) values (60 MHz, 37°C) in skin samples of subtotally (5/6th) nephrectomized
rats treated with Dotarem, Omniscan, or gadodiamide at day 4 (ie, during the 5-day injection period) and day 11 (sacrifice).
¤P , 0.001 versus Dotarem at Day 4, #P , 0.05 versus Dotarem at day 11. **P , 0.01. NS indicates non significant.
FIGURE 5. Relaxivity in vivo r1 apparent (app.) values (60 MHz, 37°C) in bone (femur epiphysis) samples of subtotally (5/6th)
nephrectomized rats treated with Dotarem (n 5 1), Omniscan (n 5 8), or gadodiamide (n 5 2) measured at day 11 (sacri-
fice). In the Dotarem-treated group, only one rat had measurable r1, as the 1/T1 2 1/T1(diamagnetic) value was less than 10%
of 1/T1(diamagnetic) in the remaining 7 rats due to the low Gd concentration.
“ionic” (in which the remaining carboxyl groups are salified with the rats treated with gadodiamide, a result consistent with the
meglumine or sodium).19 Because of their relatively low stability, literature.28,37 This underlines the harmful role of dissociated Gd
pharmaceutical solutions of some GC include a relatively large present in the Omniscan solution in the absence of the free ligand
amount of free ligand, or sodium salt of calcium complexes. These caldiamide. No macroscopic lesions were observed in the Dotarem
excipients are intended to ensure the absence of free Gd31 cations in and Omniscan groups. Virtually no histologic lesions were observed
pharmaceutical solutions for the duration of their shelf lives. An in the Dotarem group, whereas degradation of collagen fibers was
excess in free, dissociated ligand actually reduces the dissociated Gd observed in the dermis of Omniscan-treated rats. Other authors have
concentration in the pharmaceutical solution and acts like a “Gd
sponge.” The least stable agents such as Omniscan have consider- reported macroscopic lesions in rats treated with Omniscan: in 1
ably larger amounts of excess ligand (25 mmol/L of caldiamide, ie, study,28 lesions were observed by the eighth day of 5 mmol/kg daily
5% of the Omniscan concentration) than the most stable GC. There injections to SNx rats (2/4 rats involved). The (daily and cumulative)
is no excess ligand in the approved pharmaceutical formulation of doses used in Grant study28 were much higher than those used in the
Dotarem.19 present study. In another study also involving SNx rats,27 3 of the 12
The majority of cases of NSF reported in the literature to date Omniscan-treated rats developed macroscopic skin lesions starting
have been associated with administration of the nonionic, linear GC on day 5 after the last injection. The discrepancy between our data
Omniscan,32 although several reports have also concerned the ionic and these results may be explained by the early sacrifice deliberately
linear Magnevist and the nonionic linear GC OptiMARK.33–36 In the chosen in our study (to obtain sufficiently high tissue Gd concen-
present study, high morbidity and mortality were observed among trations to allow relaxometry studies). It is also noteworthy that,

even in the absence of macroscopic lesions, histologic skin lesions Relaxivities of all GC in water and plasma at various fields
(including small superficial epidermal lesions) were observed in the are available in the literature.39 The r1 relaxivity of Dotarem and
Omniscan group. Omniscan by “spiking” these GC in water, rat plasma, and the tissue
When measured at day 4, the total Gd concentration in dorsal matrices investigated in our in vitro study (skin and trabecular
skin was similar in all the groups, an effect consistent with the fact femur) were measured at the same magnetic field as our in vivo
that skin sampling was performed during the injection week. At day studies. The results obtained in both water and plasma showed good
11 (ie, at sacrifice), the Gd concentration was higher in the skin of agreement with the r1 relaxivity values published by Rohrer et al at
Omniscan-treated rats than in that of Dotarem-treated rats. The Gd the same (clinical) magnetic field.39 It has been clearly established
level in bone tissue was also higher in the Omniscan groups than in that the relaxivity of GC is higher in plasma than in water, and that
the group receiving the macrocyclic agent Dotarem. This result is relaxivity increases with macromolecular content.40,41 Our in vitro
consistent with another study.38 Sieber et al found a qualitative spiking results are fully consistent with these differences in relax-
correlation between Gd concentrations in rat skin and the develop- ivity based on the molecular composition of the biologic matrix.
ment of NSF-like skin lesions.37 They reported that the highest Gd It can be assumed that, provided the samples are processed
concentrations were measured after administration of gadodiamide under strictly identical conditions, an increase in the r1 relaxivity
(compared with Omniscan and Magnevist, a more stable, ionic value reflects the dissociation state of Gd31 that remains soluble in
linear GC). In the present study, Gd concentration at sacrifice was the extracellular space, as the r1 relaxivity values of Gd acetate in
776 nmol/g for gadodiamide (n 5 2 surviving rats), 490 6 223 water (10.4 mM21s21) and in the biologic matrices plasma and bone
nmol/g for Omniscan, and 161 6 85 nmol/g for Dotarem. A similar (15.4 and 15.8 mM21s21, respectively) are higher than those of
pattern was observed in the femur (Fig. 3). either Dotarem or Omniscan. Moreover, the possible binding of
A similar plasma clearance profile was observed for Dotarem dissociated Gd to proteins or other biomolecules should further
and Omniscan over the time windows selected in this study, which increase the apparent relaxivity value.23,42 Conversely, a decrease in
is perfectly consistent with the fact that both nonspecific GC share the r1 value would be consistent with precipitation of Gd in a form
a similar pharmacokinetic behavior.20,30 However, when taking into with little or no influence on the longitudinal relaxation rate of
account the dissociated Gd31 concentration measured by the highly protons, as demonstrated by the relaxivity values obtained for the 3
sensitive HPLC-ICP-MS technique, the Gd31 concentration mea- precipitated forms of Gd (Table 5).
sured in the plasma of Dotarem-treated rats at sacrifice was below For the first time, our relaxometry results indicate a gradual in
the LOD, but was in the micromolar range in the Omniscan-treated vivo dissociation of the linear, nonionic GC Omniscan in the skin
groups. The relatively high volume of plasma required for HPLC- and bone of renally impaired rats, with release of soluble Gd, while
ICP-MS measurements did not allow this assay to be performed at the ionic and macrocyclic GC Dotarem remained stable throughout
earlier time points. the study. These results are in line with those of plasma dissociated
We report, for the first time, in vivo dissociation of a linear Gd concentration measurement studies using HPLC-ICP-MS.
GC but not of a macrocyclic molecule. In their in vitro study, using Thermodynamic stability (which reflects the affinity of Gd for
the same measurement technique, Frenzel et al ranked GC in the its ligand) and kinetic stability (which reflects the rate at which the
following order according to their stabilities in human serum at pH equilibrium between the metal and its ligand is reached) are both
7.4 and 37°C (% of Gd release after 15 days and initial rate): essential to rigorously describe the physicochemical characteristics
nonionic linear GC . ionic linear GC . macrocyclic GC (for which
of a GC.19 The higher thermodynamic and kinetic stability associ-
values were , LOQ).21 However, because of the modalities of
ated with macrocyclic agents compared with linear GC19 has been
preparation of tissue samples, HPLC-ICP-MS cannot be used to
directly21 or indirectly43– 49 confirmed by numerous studies.
measure the dissociated Gd concentration ratio in solid tissues such
An in vivo dissociation of linear, nonionic GC has been
as skin or bone. Another approach must therefore be used. In vivo
suspected for a long time48 but has not been formally demonstrated.
relaxometry study of tissue samples is a useful tool for investigating
Numerous studies have demonstrated the presence of Gd in skin
the dissociated versus chelated state of Gd after systemic adminis-
biopsies from NSF patients, by means of sophisticated techniques.1–7
tration of GC.
However, it should be stressed that, just as these approaches, the
Paramagnetic GC induce an increase in the longitudinal and
quantitative and elementary ICP-MS technique is unable to formally
transverse relaxation rates (1/T1 and 1/T2, respectively) of solvent
nuclei.22,23 The diamagnetic and paramagnetic contributions to discriminate between chelated and dissociated Gd.
relaxation rates are additive and given by the following equation: Our data might appear to challenge the results of a recent
study which, using energy dispersive x-ray spectroscopy coupled
with scanning electron microscopy, concluded on the presence of
1/Ti(observed) 5 1/Ti(diamagnetic) 1 1/Ti(paramagnetic), insoluble tissue deposits of Gd with coassociated elements (sodium,
calcium, and phosphorus, sometimes iron or zinc) in skin biopsies of
(1) NSF patients, sampled between 2 weeks and 3 years after GC expo-
where i 5 1 or 2 and 1/T1(diamagnetic) refers to the solvent sure.50 Another study, where autopsy skin tissues from a NSF patient
relaxation rate in the absence of a paramagnetic species and 1/Ti(pa- were examined using synchrotron x-ray fluorescence microscopy and
ramagnetic) is the additional paramagnetic contribution of the GC.22 extended absorption fine structure spectroscopy, also concluded that the
Relaxivities (r1 and r2) are defined as the slope of the linear cutaneous Gd deposits found consisted of a Gd phosphate insoluble
regression generated from the solvent relaxation rates versus the material.51 Actually, both states of Gd (ie, soluble, protein-bound, and
concentration of the paramagnetic GC22: precipitated) are not mutually exclusive. It can be speculated that
following in vivo dissociation of gadodiamide and according to the
time-point and biologic (extracellular or intracellular) compartment
1/Ti(observed) 5 1/Ti(diamagnetic) 1 ri @GC#, (2) considered, Gd may be present in both the states.
where @GC# is the concentration of the Gd chelate and Possible ligands in plasma include phospholipids, amino
acids, peptides, nucleotides, proteins, and many other compounds
present in blood carrying oxygen-donor atoms.52 The present study
ri 5 @1/Ti(observed) 2 1/Ti(diamagnetic)#/@GC# (3) was not designed to investigate in vivo biospeciation. This issue is

obviously crucial, as it determines the role of Gd in the mechanism 7. High WA, Ranville JF, Brown M, et al. Gadolinium deposition in nephro-
of NSF. genic systemic fibrosis: an examination of tissue using synchrotron x-ray
fluorescence spectroscopy. J Am Acad Dermatol. 2010;62:38 – 44.
Provided samples are stored under good conditions and the
8. High WA, Ayers RA, Chandler J, et al. Gadolinium is detectable within the
Gd concentration is sufficient, the relaxometry approach presented tissue of patients with nephrogenic systemic fibrosis. J Am Acad Dermatol.
here could be used in human biopsy samples from NSF or control 2007;56:21–26.
patients to investigate the dissociated versus chelated and soluble 9. Swaminathan S, High WA, Ranville J, et al. Cardiac and vascular metal
versus precipitated forms of Gd. deposition with high mortality in nephrogenic systemic fibrosis. Kidney Int.
Our data do not provide any information on the compartment 2008;73:1413–1418.
(intracellular or extracellular) in which the dissociation of Omniscan 10. Idée JM, Port M, Medina C, et al. Possible involvement of gadolinium
occurs. In vitro studies suggest that the incubation of Omniscan with chelates in the pathophysiology of nephrogenic systemic fibrosis: a critical
review. Toxicology. 2008;248:77– 88.
cultured cells was associated with a shift in the dissociation equi-
11. Morcos SK. Nephrogenic systemic fibrosis following the administration of
librium that, in turn, resulted in a net transfer of Gd31 ions onto the extracellular gadolinium-based contrast agents: is the stability of the contrast
cell membrane followed by their internalization. Furthermore, re- agent molecule an important factor in the pathogenesis of this condition? Br J
moval of phosphate from the culture medium was associated with a Radiol. 2007;80:73–76.
10-fold decrease in Gd cellular uptake, suggesting a transmetallation 12. Perazella MA. Tissue deposition of gadolinium and development of NSF: a
process.45 However, this does not necessarily imply the formation of convergence of factors. Semin Dial. 2008;21:150 –154.
insoluble GdPO4. The addition of 10 mM phosphate to human serum 13. Steger-Hartmann T, Raschke M, Riefke B, et al. The involvement of pro-
accelerates the release of soluble Gd31 from gadodiamide.21 inflammatory cytokines in nephrogenic systemic fibrosis. A mechanistic
hypothesis based on preclinical results from a rat model treated with gado-
Although Omniscan was shown to dissociate in vivo, unlike diamide. Exp Toxicol Pathol. 2009;61:537–552.
the macrocyclic GC Dotarem, the exact role of Gd in the mechanism 14. Varani J, DaSilva M, Warner RL, et al. Effects of gadolinium-based magnetic
of NSF remains speculative. Numerous prospective28,29,37,38 and resonance imaging contrast agents on human skin in organ culture and human
retrospective53 in vivo preclinical studies have been performed to skin fibroblasts. Invest Radiol. 2009;44:74 – 81.
better understand the pathophysiology of the disease. Soluble Gd31 15. Fu LJ, Li JX, Yang XG, et al. Gadolinium-promoted cell cycle progression
(GdCl3) (2– 60 mM) promotes mouse embryo fibroblast growth with enhanced S-phase entry via activation of both ERK and PI3K signaling
pathways in NIH 3T3 cells. J Biol Inorg Chem. 2009;14:219 –227.
through activation of both ERK (extracellular signal-regulated ki-
nase) and PI3K (phosphatidylinositol 3-kinase) pathways.15 A pro- 16. Stratta P, Canavese C, Aime S. Gadolinium-enhanced magnetic resonance
imaging, renal failure and nephrogenic systemic fibrosis/nephrogenic fibros-
liferative effect of GdCl3 (5 and 10 mM) has also been shown on ing dermopathy. Curr Med Chem. 2008;15:1229 –1235.
human dermal fibroblasts, an effect inhibited by the free ligand 17. Wermuth PJ, Del Galdo F, Jiménez SA. Induction of the expression of profibrotic
DTPA (diethylenetriamine pentaacetic acid).14 DaSilva et al re- cytokines and growth factors in normal human peripheral blood monocytes by
cently reported that Omniscan induced human fibroblast prolifera- gadolinium contrast agents. Arthritis Rheum. 2009;60:1508 –1518.
tion and directly stimulated hyaluronan production. Furthermore, 18. Newton BB, Jiménez SA. Mechanism of NSF: new evidence challenging the
this GC was found to alter the matrix metalloproteinase-1/tissue prevailing theory. J Magn Reson Imaging. 2009;30:1277–1283.
inhibitor of metalloproteinases-1 system, which is responsible for 19. Port M, Idée JM, Medina C, et al. Efficiency, thermodynamic and kinetic
regulating collagen turnover in the skin.54 Moreover, another study stability of marketed gadolinium chelates and their possible clinical conse-
quences: a critical review. Biometals. 2008;21:469 – 490.
from the same team demonstrated that Gd31 induced the proliferation
20. Idée JM, Port M, Dencausse A, et al. Involvement of gadolinium chelates in
of human dermal fibroblasts, an effect modulated through the platelet- the mechanism of nephrogenic systemic fibrosis: an update. Radiol Clin
derived growth factor receptor-associated signaling pathway.55 North Am. 2009;47:855– 869.
Like other lanthanides, Gd may also interfere with fibrillo- 21. Frenzel T, Lengsfeld P, Schirmer H, et al. Stability of gadolinium-based
genesis by attaching to the helical part of the collagen molecule.16 magnetic resonance imaging contrast agents in human serum at 37°C. Invest
However, as indicated previously, the presence of insoluble Gd (in Radiol. 2008;73:817– 828.
the form of phosphate or other salts) in the skin is very likely. It has 22. Lauffer RB. Paramagnetic metal complexes as water proton relaxation agents
been proposed that Gd phosphate may trigger pro-inflammatory for NMR imaging: theory and design. Chem Rev. 1987;87:901–927.
events.6,11–13 Undoubtedly, additional studies are needed to eluci- 23. Caravan P, Ellison JJ, McMurry TJ, et al. Gadolinium(III) chelates as MRI
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In summary, our results are strongly indicative of gradual in vivo 24. Chamberlain RM, Shirley DG. Time course of the renal functional response
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ORIGINAL ARTICLE
Clinical, Biological, and Skin Histopathologic Effects of Ionic

Macrocyclic and Nonionic Linear Gadolinium Chelates in a Rat
Model of Nephrogenic Systemic Fibrosis
Nathalie Fretellier, MS,* Jean-Marc Idée, PharmD,* Sylviane Guerret, PhD,† Claire Hollenbeck, BS,*
Daniel Hartmann, PharmD,‡ Walter González, PhD,* Caroline Robic, PhD,* Marc Port, PhD,*
and Claire Corot, PharmD*
may develop, with some patients progressively becoming wheel-

Objective: The purpose of this study was to compare the clinical,
chair-dependent. Patients often complain of pruritus, causalgia, and
pathologic, and biochemical effects of repeated administrations of ionic
sharp pains. The distal extremities are the most common site of
macrocyclic or nonionic linear gadolinium chelates (GC) in rats with
involvement, followed by the trunk.1,2
impaired renal function.
A link between the administration of gadolinium chelates
Material and Methods: Rats submitted to subtotal nephrectomy were
(GC) for magnetic resonance imaging procedure and the develop-
allocated to single injections of 2.5 mmol/kg of gadodiamide (nonionic linear
ment of NSF is now generally accepted.3
chelate), nonformulated gadodiamide (ie, without the free ligand caldia-
Basically, 2 structurally distinct categories of GC are
mide), gadoterate (ionic macrocyclic chelate), or saline for 5 consecutive
currently marketed: (a) “macrocyclic” chelates (gadoterate, ga-
days. Blinded semi-quantitative histopathologic and immunohistochemical
doteridol, or gadobutrol) in which the Gd31 ion is “caged” in the
examinations of the skin were performed, as well as clinical, hematological,
preorganized cavity of the ligand, and (b) “linear” (or “open
and biochemical follow-up. Rats were killed at day 11. Long-term (up to day
chain”) chelates (gadopentetate, gadobenate, gadodiamide, ga-
32) follow-up of rats was also performed in an auxiliary study.
doversetamide, gadofosveset, and gadoxetate). GC can also be
Results: Epidermal lesions (ulcerations and scabs) were found in 4 of the
either “nonionic” (in which the number of carboxyl groups is
10 rats treated with nonformulated gadodiamide. Two rats survived the
reduced to 3, neutralizing the 3 positive charges of Gd31) or
study period. Inflammatory signs were observed in this group. No
clinical, hematological, or biochemical signs were observed in the saline
“ionic” (where the remaining carboxyl groups are salified with
and gadoterate- or gadodiamide-treated groups. Plasma fibroblast growth
meglumine or sodium).4 GC differ in terms of the molecular
factor-23 levels were significantly higher in the gadodiamide group than
stability of their solutions. High kinetic stability provided by the
in the gadoterate group (day 11). Decreased plasma transferrin-bound
macrocyclic structure combined with high thermodynamic stabil-
iron levels were measured in the nonformulated gadodiamide group.
ity minimizes the amount of free Gd31 that can be gradually
Histologic lesions were in the range: nonformulated gadodiamide (super-
released from the parent chelate.4 To reduce the risk of gradual
ficial epidermal lesions, inflammation, necrosis, and increased cellularity
Gd31 release in the pharmaceutical solution during shelf-life,
in papillary dermis) . gadodiamide (small superficial epidermal lesions
several GC, especially low stability GC such as gadodiamide, are
and signs of degradation of collagen fibers in the dermis) . gadoterate
formulated with a free ligand.4
(very few pathologic lesions, similar to control rats). To date, the majority of cases of NSF reported in the literature
Conclusions: Repeated administration of the nonionic linear GC gadodia- have been associated with administration of the nonionic, linear GC
mide to renally impaired rats is associated with more severe histologic gadodiamide,5 although several reports have also concerned other
lesions and higher FGF-23 plasma levels than the macrocyclic GC GC, notably the ionic linear gadopentetate and the nonionic linear
gadoterate. GC gadoversetamide.6 –9 No unconfounded case report involving the
ionic macrocyclic GC gadoterate has been published so far. Despite
Key Words: nephrogenic systemic fibrosis, gadolinium chelates, of active research, the mechanism of NSF has not, as yet, been
gadodiamide, gadoterate, renal impairment, fibroblast growth factor-23 definitely determined.10 Several authors have suggested that in vivo
(Invest Radiol 2011;46: 85–93) dechelation of GC may trigger profibrotic pathways, in the presence
of risk factors,11,12 thus underscoring the importance of the thermo-
dynamic and kinetic stabilities. However, on the basis of in vitro
data, the pathophysiologic relevance of these physicochemical char-
N ephrogenic systemic fibrosis (NSF) is a scleroderma-like dis-
ease comprising extensive thickening and hardening of the skin
associated with skin-colored to erythematous papules that coalesce
acteristics in NSF has been disputed.13
Selection of appropriate animal models is an important chal-
lenge not only to provide a better understanding of the mechanism
into erythematous to brawny plaques with a peau d’orange appear- of the disease, but also to allow early screening of candidate GC
ance, always occurring in patients with severe or end-stage renal under preclinical development. As severe chronic kidney disease is
failure. Nodules are sometimes also described. Joint contractures the most obvious factor associated with NSF, it seemed appropriate
to investigate rats with impaired renal function. Subtotal (5/6)
nephrectomy has been shown to delay clearance of GC in rats, an
Received May 20, 2010; accepted for publication, after revision, July 10, 2010. effect associated with a higher concentration of gadolinium in the
From the *Guerbet, Research Division, Aulnay-Sous-Bois, France; †Novotec,
Lyon, France; and ‡School of Pharmacy, USPS 2007.03.135, University skin of rats that received the nonionic linear GC gadodiamide and
Claude Bernard Lyon I, Lyon, France. gadoversetamide when compared with the ionic linear GC gado-
Reprints: Jean-Marc Idée, PharmD, MS, Research Division, BP 57400, 95943 pentetate. The lowest values were found in rats treated with a
Roissy Charles de Gaulle Cedex, France. E-mail: jean-marc.idee@
guerbet-group.com.
macrocyclic GC (gadobutrol).14
Copyright © 2011 by Lippincott Williams & Wilkins The aim of the present study was to compare an ionic,
ISSN: 0020-9996/11/4602-0085 macrocyclic GC (gadoterate) (high thermodynamic and kinetic sta-
Investigative Radiology • Volume 46, Number 2, February 2011 www.investigativeradiology.com | 85

Fretellier et al Investigative Radiology • Volume 46, Number 2, February 2011
bilities) with the nonionic, linear GC gadodiamide (low thermody- tomat (Melet-Schloesing, Osny, France). The blood levels of total
namic and kinetic stabilities), the latter both formulated and nonfor- calcium, phosphorus, transferrin-bound iron, aspartate aminotrans-
mulated with free ligand caldiamide, with respect to their clinical, ferase (AST), alanine aminotransferase (ALT), creatinine (Vitros-II
histopathologic (both short- and long-term), biochemical, and he- auto-analyzer), sodium, chloride, and potassium (direct potentiom-
matological effects, in rats with impaired renal function. etry, Vitros-350 autoanalyzer, OrthoClinical Diagnostics Inc, Issy-
les-Moulineaux, France) were also measured. The biologic param-
MATERIALS AND METHODS eters were measured on days 0, 3, 9, and 11. Blood samples were
taken from the sublingual vein.15 All assays were performed in
Animal Model duplicate.
All studies were carried out on male, Wistar rats from CERJ In addition, the C-reactive protein levels were measured by
(Centre d’Elevage René Janvier, Le Genest Saint-Isle, France), aged enzyme-linked immunosorbent assay (ELISA) (United States
6 weeks and weighing 235 6 23 g. Biologic, Swampscott, MA) before the first injection and on day
The animals underwent subtotal (5/6) nephrectomy (SNx): 3. Plasma levels of the cytokines interleukin-1-b (IL1-b), mono-
the right kidney was exposed through a flank incision, the adrenal cytes chemotactic protein-1 (MCP-1), tumor necrosis factor-a
gland was separated from the upper pole, and the kidney was (TNFa), and transforming growth factor b1 (TGFb1) were mea-
decapsulated. The renal pedicle was ligated and the right kidney was sured by ELISA (Bender MedSystems, Vienna, Austria) on days
removed. Subsequently, the left kidney was decapsulated and the 3 and 9. Plasma levels of fibroblast growth factor 23 (FGF-23) at
adrenal gland was separated from the upper pole. Ligatures were day 11 were measured by ELISA (Kainos Laboratories, Tokyo,
placed around the upper and lower poles and the poles were excised. Japan). The FGF-23 levels of subtotal nephrectomized rats re-
Animals were anesthetized with ketamine and xylazine. An intrave- ceiving a high phosphate diet (1.0%) and sham-operated rats
nous injection of 1.0 mL/kg of saline was performed on completion receiving a normal diet were also measured at day 35 postsurgery
of surgery to compensate for blood loss. The surgical procedure was (plasma phosphorus levels 3.9 (n 5 2) and 2.3 6 0.4 mmol/L,
carried out at the CERJ laboratory. respectively).
The animals were housed 1 per cage at an ambient tempera-
ture of 22°C 6 2°C, hygrometry of 55% 6 10%, with 12/12 Macroscopic Skin Findings and Histology
light/dark cycles. Animals had free access to water and standard The rats were checked for macroscopic skin changes before
animal chow (reference A04, SAFE, Augy, France) containing (per the first injection and then daily throughout the study. On day 4, the
kilogram) 5.9 g phosphorus, 8.3 g calcium, 2.5 g sodium, 6.7 g back of the animals were shaved for better visualization of potential
potassium, 2 g magnesium, 85 mg zinc, and 240 mg iron. The lesions. The backs and abdomens were shaved again at completion
protein concentration was 16.1%. of the study. Skin samples (2 cm2) were taken from the backs of
All experimental procedures were carried out according to animals (normal skin and lesions). The skin samples were fixed in
French rules and in compliance with the European Economic Com- 4% neutral buffered formalin. After routine dehydration, the sam-
munity Directive (86/609/EC) on animal welfare. The protocol was ples were embedded in paraffin, sectioned (5 mm thickness) for
approved by the local ethics committee. hematoxylin-eosin-saffron for topographical analysis, picrosirius red
to detect the presence of collagen, and Alcian blue for acidic
Comparative Gadoterate Versus Gadodiamide mucopolysaccharides. Immunohistochemical staining was per-
Study formed to detect CD34 (cluster of differentiation 34) and TGFb1
Thirty-three male, Wistar subnephrectomized rats from positive cells using anti-CD34 goat antibody (diluted to 1:1000,
CERJ, aged 6 weeks and weighing 232 6 26 g were allocated to a R&D Systems, Minneapolis, MN) and anti-TGFb1 rabbit antibody
daily treatment with either saline (5.0 mL/kg), 0.5 M meglumine (diluted to 1:250, GeneTex, San Antonio, TX) using ImmPRESS
gadoterate (Dotarem, Guerbet, Villepinte, France, batch 8GD051B), polymerized reporter peroxidase staining (ImmPRESS anti-goat Ig
0.5 M gadodiamide (Omniscan, GE Healthcare, Chalfont St Giles, (peroxidase) kit (MP 7405) and ImmPRESS anti-rabbit Ig (peroxi-
United Kingdom batch 10758384), or nonformulated gadodiamide dase) kit (MP 7401), respectively (Vector Laboratories Burlingame,
(ie, without the 25 mM excess ligand caldiamide present in the CA).
commercial solution of gadodiamide) (0.495 M, synthesized at the Histopathologic examinations were performed in a blinded
Guerbet Research Department). The rats received saline or 2.5 manner by a certified histopathologist (S.G.) in accordance with the
mmol/kg of each GC intravenously via the tail vein at a rate of 1.0 guidelines of the Society of Toxicologic Pathology.16
mL/min, daily for 5 consecutive days, starting 10 days after subtotal Pathologic lesions, especially the distribution of thin or
nephrectomy. This period was defined after a preliminary study thick collagen fibers in the extracellular matrix (ECM) and CD34
where 10 SNx rats were compared with 4 control, sham-operated and TGFb1 positive cells, were evaluated semi-qualitatively
animals. The creatinine clearance of the SNx rats was found to be using a 5-point severity grading scale ranging from 0 (absent) to
significantly decreased (266%) from the first measurement (day 7) 111 (very severe) for each animal. In the long-term study, the
when compared with sham-operated rats (P , 0.05). The creatinine expression of CD34 and TGFb1 was investigated by calculating,
clearance remained stable in the SNx group all over the study for each rat, the percentage of positive cells per field (5 fields/rat)
(0.38 – 0.48 mL/min/100 g). Creatinine clearance was 1.0 to 1.2 at day 4 and 32.
mL/min/100 g in the sham-operated control group.
The rats were killed (exsanguinations under isoflurane anes- Long-Term Study
thesia) 11 days after the first administration. To investigate possible long-term histopathologic effects, 10
All injections and blood samples were performed under male Wistar rats, from CERJ, aged 6 weeks and weighing 222 6
isoflurane/oxygen (3.5% induction, then 2.5%, 1 L/min oxygen) 17 g, were injected intravenously with 5 consecutive once-daily
anesthesia. doses of saline (2 rats) or 2.5 mmol Gd/kg bodyweight of gadoterate
(4 rats) and gadodiamide (4 rats), starting 10 days after subtotal
Biochemistry and Hematology nephrectomy.
Hematology (erythrocyte, leukocyte and platelet counts, he- Skin biopsies were performed at day 4, 11, 18, and 25 after
matocrit, hemoglobin concentration) was studied using a MS4 au- first injection, under isoflurane/oxygen anesthesia with a biopsy

Investigative Radiology • Volume 46, Number 2, February 2011 Effects of Gadolinium Chelates in Renally Impaired Rats
TABLE 1. Observed Macroscopic Skin Lesions and Other Clinical Symptoms After Treatment With Gadoterate, Gadodiamide,
Nonformulated Gadodiamide (5 3 2.5 mmol/kg Daily), or Saline (5 3 5.0 mL/kg Daily)
No. Rats per No. Rats Alive at Completion No. Rats With Skin
Compounds Group of the Study (Day 11) Lesions Other Symptoms
Saline 7 7 0 —
Gadoterate (Dotarem) 8 8 0 —
Gadodiamide (Omniscan) 8 8 0 —
Nonformulated gadodiamide 10 2 (5 rats euthanized for ethical 4 (starting at day 5) (scab Prostration, piloerection, loss
reasons and 3 rats found formation and ulceration) of bodyweight
dead @day 3, 7, and 9#)
injection) for 2 rats and day 9 for 2 rats (Fig. 1). Eight rats were
either found dead or had to be killed for ethical reasons because
of the severity of the skin lesions and loss of bodyweight. No
scratching was observed.
No macroscopic skin lesions were observed in the rats
treated with saline, gadoterate, or gadodiamide. No significant
difference in bodyweight changes was observed between these
groups. However, the 2 surviving rats treated with nonformulated
gadodiamide experienced a loss in body weight (mean values:
day 1: 287.8 g, day 11: 270.6 g).
Semi-quantitative histopathologic data are shown in
Table 2, and typical lesions are illustrated in Figure 2. Very few
histopathologic lesions were reported in the gadoterate-treated
group or in the saline controls. The dermis presented constant
thickness and low cellularity. ECM was dense underneath the
epidermis and homogenous in the dermis (saline) and was ho-
mogenous in the gadoterate group. Small superficial epidermal
lesions and degradation of collagen fibers in the dermis with
disorganization of the ECM were observed in the gadodiamide
group. The ECM was dense underneath the epidermis and looser
FIGURE 1. Typical abdominal skin lesions of a rat treated in the deep dermis. Numerous superficial epidermal lesions,
with nonformulated gadodiamide (day 9). inflammation, necrosis, and increased in cellularity, as well as
large areas of complete degradation of the ECM in the dermis
were observed in the nonformulated gadodiamide group. CD34 or
TGFb1 positive immunolabeling was observed on spindle-shaped
punch (6-mm diameter, Labonord, Templemars, France) and the
wounds were then sutured. The sample was fixed in 4% neutral cells (fibroblast-like) and dendrocytes in the dermis. However,
buffered formalin for histology. the dermal distribution of positive CD34 and TGFb1 cells was
The rats were necropsied (exsanguination under isoflurane heterogeneous.
anesthesia) 32 days after the first administration.
Biochemistry and Hematology
Statistical Analysis No significant differences were observed between groups
Data are expressed as mean 6 standard deviation. Statistical for plasma levels of sodium, potassium, chloride, ALT, and AST
analyses were carried out using GraphPad Prism (GraphPad Soft- levels. Plasma phosphorus levels were decreased in gadodiamide-
ware Inc, San Diego, CA) or SAS (Statistical Analysis Software, treated rats compared with controls at day 9 (1.6 6 0.2 mmol/L
Cary, NC). For each study, the homogeneity between groups was and 2.2 6 0.3 mmol/L, respectively, P , 0.001) and day 11 (1.6
verified by analysis of variance. The effects of the test-solutions on 6 0.2 mmol/L and 2.0 6 0.2 mmol/L, respectively, P , 0.05). At
bodyweight, biochemical, and hematological parameters were com- day 9, the plasma phosphorus level was also significantly lower
pared by analysis of variance with repeated measures with calcula- in the gadodiamide group than that in the gadoterate group (1.6
tion of the time 3 product interaction.17 A P # 0.05 was considered 6 0.2 mmol/L vs. 2.0 6 0.2 mmol/L, respectively, P , 0.05). A
to indicate a significant difference. decrease in plasma iron levels was observed in the group receiv-
ing nonformulated gadodiamide (day 0: 58.6 6 16.2 mmol/L @n 5
RESULTS 10 rats#, day 11: 28.1 mmol/L @n 5 2 rats#) while no changes were
observed in the other groups.
Comparative Gadoterate Versus Gadodiamide
No changes in hematology parameters were observed, apart
Study (Short-Term Study) from a dramatic increase in the leukocyte count in the nonformu-
Macroscopic and Microscopic Findings lated gadodiamide group (at day 0: 24.1 6 1.9 3 103 cells/mm3, at
Clinical data are shown in Table 1. Skin lesions (ulcer- day 11: 57.2 3 103 cells/mm3). The differential white cell count was
ations and scabs) were found in 4 of the 10 rats treated with granulocytes: 42.4% 6 1.7%, lymphocytes: 53.1% 6 1.7%, and
nonformulated gadodiamide in the neck, back, and abdominal monocytes: 4.5% 6 0.2% on day 0; and granulocytes: 71.3%,
skin, observed for the first time on day 5 (ie, after the first lymphocytes: 26.3%, and monocytes: 2.5% on day 11 (2 rats).

TABLE 2. Semiquantitative Histopathologic Lesions

Dermis
Epidermis Thin Thick
Local Mast Fibres Fibres Wide
Lesion Necrosis Cellularity Inflammation Necrosis Fibroblasts Cells Dendrocytes ECM ECM Clefts Mucus TGFb1 CD34
Saline 7 (2) 7 (2) 7 (1) 6 (2) 7 (2) 7 (1) 7 (6) 4 (6) 2 (11) 5 (6) 1 (6) 2(2) 3 (1) 2 (1) 1
(11) 1 (1) 3 (1) 5 (111) 2 (1) 6 (1) 5(6) 1 (11) 5 (11) 5
(1) 3 (111) 1
Gadoterate 8 (2) 7 (2) 7 (1) 8 (2) 7 (2) 8 (6) 3 (1) 6 (1) 8 (1) 6 (6) 2 (1) 6 (2) 2 (1) 5 (1) 2
(6) 1 (6) 1 (1) 1 (1) 5 (11) 2 (11) 2 (1) 6 (11) 2 (6) 5 (11) 3 (11) 5
(1) 1 (111) 1
Gadodiamide 8 (2) 6 (2) 8 (1) 8 (2) 6 (2) 8 (1) 8 (1) 7 (1) 8 (11) 8 (1) 2 (1) 7 (6) 5 (1) 5 (1) 2
(6) 2 (1) 2 (11) 1 (11) 6 (11) 1 (1) 2 (11) 3 (11) 6
(11) 1
Nonformulated 2 (11) 2 (11) 2 (11) 2 (11) 2 (11) 2 (6) 2 (1) 2 (1) 2 (1) 2 (11) 2 (1) 2 (6) 1 (11) 2 (1) 2
Gadodiamide (1) 1
2 indicates absence; 6, mild; 1, moderate; 11, severe; 111, very severe.
FIGURE 2. Histologic skin lesions in treated, renally impaired rats: short-term study. Hematoxylin and eosin staining (A–C) to
analyze cellular lesions and picrosirius red staining (D–I) to detect ECM modifications (320 magnification). After saline treat-
ment (A, D, G): epidermis (E) thickness was constant; the dermis (D) was weakly cellular, and the ECM was homogeneous.
After gadoterate treatment (B, E, H): several zones of epidermis were sometimes thickened; in the dermis only the ECM was
slightly modified, showing slight condensation (3) under the dermoepidermal junction. Gadodiamide treatment (C, F, I) in-
duced local epidermal necrosis and some inflammatory cells (‹) in the papillary dermis (or superficial); ECM was heteroge-
neous, showing small clusters of merged collagen fibers (E) or locally dissociated collagen fibers (w).
No significant variation of plasma C-reactive protein was 9 (n 5 4 rats), plasma IL1-b levels were significantly higher (P
observed between test groups at day 3 (data not shown). , 0.05) in this group than in the gadodiamide group (Fig. 3A). A
Plasma IL1-b levels were significantly higher (P , 0.01) significant increase in plasma MCP-1 levels was also observed in the
in the nonformulated gadodiamide group than in the saline, group receiving nonformulated gadodiamide, both during the injection
gadoterate, and gadodiamide groups at day 3 (n 5 8 rats). At day period (day 3; P , 0.001) and at day 9 (P , 0.001) (Fig. 3B). No effect

FIGURE 3. A, Plasma IL-1 b levels in subtotal (5/6) nephrectomized rats at day 3 (during injection period) and day 9. B,
Plasma MCP-1 levels in subtotal (5/6) nephrectomized rats at day 3 (during injection period) and day 9. The rats were intra-
venously injected on 5 consecutive days with 2.5 mmol/kg/d.
FIGURE 4. Plasma FGF-23 levels in subtotal

(5/6) nephrectomized rats at day 11 (killed).
The rats were intravenously injected on 5 con-
secutive days with 2.5 mmol/kg/d. §P , 0.05
versus saline, #P , 0.01 versus gadoterate.
The histologic lesions found at day 4 and 32 are summarized

TABLE 3. Histopathologic Lesions of the Dorsal Skin in Table 3 and typical lesions are illustrated in Figure 5. More
Observed in the Long-Term Study lesions were found on day 32 than on day 4 and more lesions were
Histopathological Lesions on Rat Dorsal found in the gadodiamide-treated group than in the gadoterate-
Skin at treated group. In particular, locally thickened epithelium associated
with some inflammatory foci and small clusters of damaged collagen
Treatment No. Rats Day 4 Day 32
bundles were found in 3 of 4 gadodiamide-treated rats.
Saline 2 N52 SN 5 2 Neither gadoterate nor gadodiamide had any effect on TGFb1
Gadoterate 4 N 5 3, SN 5 1 SN 5 4 or CD34 immunostaining compared with the control group, regard-
Gadodiamide 4 N 5 1, SN 5 2, 1 5 1 6 5 1, 1 5 3 less the time-point considered. Intense staining was observed at both
time-points in the control group, especially for CD34 (Table 4).
N indicates normal; SN, subnormal (rare, small areas of altered sub-epidermal
dermis, minimal inflammatory reaction); 6, locally thickened epithelium associated
with several isolated necrotic cells, heterogeneous dermal matrix (presence of both
dense and loose collagen bundles); 1, locally thickened epithelium associated with DISCUSSION
some macrophage foci and small inflammatory foci, small clusters of damaged collagen
bundles.
Despite active research, the mechanism of NSF remains
debated as the animal model must be adapted to ensure optimal
clinical relevance. To achieve this objective, this study was con-
ducted in subtotally nephrectomized rats; therefore, presenting sig-
of the test compounds was observed on TGFb1 and TNFa plasma nificantly reduced renal function. The SNx rat model used in this
levels, regardless of treatment and time-point (data not shown). study is similar to that selected by other teams.14,18,19 At 2.5
Higher FGF-23 levels were found in SNx rats receiving a mmol/kg, the dose of GC used in rats was considerably higher, per
high-phosphate diet (1370 pg/mL, n 5 2) compared with sham- unit bodyweight, than the dose range of 0.1 to 0.3 mmol/kg,
operated rats (235 6 91 pg/mL, n 5 6). Plasma FGF-23 levels were classically used for magnetic resonance imaging contrast procedures
higher in the gadodiamide treated group than in the saline (P , 0.05) in patients.20 However, this dose was considered to be appropriate
and gadoterate groups (P , 0.01) (Fig. 4). for chronic studies in rats for 2 reasons: first, comparative drug doses
between species should be normalized to body surface area rather
Comparative Gadoterate Versus Gadodiamide than body weight. The Food and Drug Administration recommends
Study (Long-Term Study) the use of a 6-fold increase to obtain a human equivalent drug dose
No macroscopic skin lesions were found in the rats treated for rats.21 On this basis, a dose of 0.4 mmol/kg GC in man, would
with saline, gadoterate, or gadodiamide. No difference in body- be equivalent to 2.5 mmol/kg in rats. Second, the excretion half-life
weight changes was observed between groups (data not shown). of GC in rats (around 20 minutes14,22) is markedly shorter than in

FIGURE 5. Histologic skin lesions in treated, renally impaired rats: long-term study (320 magnification). The long-term study
mainly showed changes of the ECM. Hematoxylin and eosin staining (A–C) showed some clusters of damaged collagen bun-
dles (circled) regardless of the treatment. TGFb1 (D–F) detection in papillary dermis was similar in the 3 groups and was only
observed in some fibroblasts and dendritic cells; note the increased number of these TGFb1 positive cells (3) in the reticular
dermis. Anti-CD34 immunolabeling revealed a general tendency toward more positive cells (fibroblast-like) in the papillary
dermis. The cellular CD341 (3) staining was weak in saline and gadoterate-treated rats, (G, H) and increased in gadodia-
mide-treated rats (I).
with inflammatory signs and skin lesions, was observed in this

TABLE 4. Percentage of CD341 and TGFb11 Cells (Mean group. This result is consistent with the results of other studies in
of 5 Fields/Rat) at Day 4 at Day 32 (Long-Term Study) rats with normal or impaired renal function.18,24 Because of its low
% of Cells Positive for intrinsic stability, the commercial solution of gadodiamide contains
25 mmol/L of caldiamide to chelate gadolinium that may be released
CD34 TGFb1
during shelf-life.23 Therefore, it is likely that dissociated gadolinium
Treatment No. Rats Day 4 Day 32 Day 4 Day 32 released in the vial during shelf-life of the test-solution triggered the
Saline 2 76.0 54.3 18.9 16.7 systemic toxicity observed in this study. It has been suggested18 that
excessive scratching caused by pruritus may cause the skin lesions
Gadoterate 4 55.0 63.8 19.8 14.5
induced by gadodiamide. It is noteworthy that, under the present
Gadodiamide 4 53.4 64.2 23.3 14.6
study conditions, no scratching was observed, despite the treatment.
Furthermore, in contrast with another study,18 no increase in dermal
mast cells was observed in gadodiamide-treated rats compared with
man (1.2–2 hours23), reducing its tissue exposure and supporting the rats treated with gadoterate (Table 2).
use of larger doses of GC in rats when calculated per unit body Visible skin lesions following administration of formulated or
weight. The mean excretion half-life of gadodiamide (2.5 mmol/kg, nonformulated gadodiamide have been reported in several stud-
single injection) in SNx rats was 58.7 6 13.3 minutes versus 19.9 6 ies,18,24 including the 21-day subchronic toxicity study of the
2.3 minutes in non-SNx rats.14 formulated solution in rats.25 It has been suggested that this effect
In a preliminary study, the creatinine clearance of SNx rats could be the consequence of caldiamide-induced zinc deficiency.23
remained stable throughout the observation period, for 61 days after Bullous pustular dermatitis, alopecia, rough skin, hypogonadism,
surgery, and was reduced by around 60% compared with sham- and oligospermia have been reported in patients with zinc defici-
operated rats. On the basis of this result, the injection time for the ency.26 However, this hypothesis is challenged by the fact that (a) a
comparative study was defined as the 10th day after surgery. similar effect was observed in rats receiving nonformulated gadodia-
Macroscopic skin lesions were detected in 4 of the 10 rats that mide, as in our study,18,24 (b) no reduction in zinc concentrations
received nonformulated gadodiamide. A high mortality, associated was detected in gadodiamide-treated rats,17 and (c) coadministration

or zinc depletion does not protect rats against gadodiamide-induced treated rats, although this effect was less intense than that observed
skin lesions.27 Furthermore, the thermodynamic stability constants for CD34. Neither GC affected the TGFb1 staining, regardless of the
are not in favor of a transmetallation phenomenon between zinc and time-point considered. TGFb has been reported to induce down-
gadolinium (log Ktherm of Zn-DTPA-BMA 5 12.0 and log Ktherm of regulation of CD34 expression by fibrocytes.36 The colocalization of
Gd-DTPA-BMA 5 16.9).28 Changes in zinc homeostasis following TGFb1 and CD34 expression was not formally investigated in our
administration of high doses of gadodiamide are therefore unlikely study but these markers were probably expressed by the same
to cause skin ulcerations. In contrast, a link has been reported (fibroblast-like) cells.
between high gadolinium concentration in the skin and histologic Nonformulated gadodiamide-induced release of chemokine
skin lesions.14 MCP-1, at both day 3 and 9. This is consistent with another study38
The clinical relevance of histologic data from rat studies in which chemokines MCP-1 and MCP-3 were significantly in-
remains a debated topic.18,23,29,30 As underlined by Sieber et al,29 creased 6 hours after the first administration of this compound to rats
differences in the anatomic structure of the skin in humans and rats with normal renal function. MCP-1 has been identified as a profi-
may make the clinical relevance of preclinical studies somewhat brotic mediator.35,39 Plasma MCP-1 levels are increased in patients
hazardous. Furthermore, not all pathologic features of human NSF with systemic fibrosis.40 Increased MCP-1 expression has also been
have been investigated in rat studies.23 Finally, microscopic dermal observed in inflammatory kidney diseases.41 Nonformulated ga-
abnormalities could be considered to be more clinically relevant dodiamide has also been reported to release other cytokines in rats.38
than macroscopic epidermal lesions. However, no changes of plasma TGFb1 and TNFa levels were
Very few histopathologic lesions were observed in the gado- observed under the present study conditions. To our knowledge, the
terate group whereas degradation of collagen fibers was observed in effect of GC on circulating TGFb1 levels has never been previously
the dermis of gadodiamide-treated rats (greater number of rats with investigated. Intense release of IL1b was observed in the nonfor-
thick, disorganized fibers) but no macroscopic epidermal lesions mulated gadodiamide-treated group, at day 3, consistent with a
were observed (Tables 1 and 2). This effect was confirmed in the systemic proinflammatory effect of this compound.
long-term study in which time-dependent inflammatory foci and Decreased plasma transferring-bound iron levels were mea-
damaged collagen bundles were observed in the gadodiamide group sured in rats receiving the nonformulated gadodiamide. A transmet-
but not in the gadoterate group (more lesions at day 32 than at day allation phenomenon23,28 between Gd31 (associated with its ligand
4). It has been speculated that soluble gadolinium released from less DTPA-BMA) and Fe31 (bound to transferrin, Tf) may be proposed,
stable GC may bind to collagen molecules, interfering with fibril- based on the more favorable thermodynamic constant of Fe-DTPA-
logenesis.31 Recent studies have clearly shown that gadodiamide BMA compared with that of gadodiamide (or Gd-DTPA-BMA) (log
interferes with collagen turnover.32,33 This might lead to the abnor- Ktherm values of 21.9 and 16.9, respectively):
mal distribution of collagen bundles observed in our study in the
gadodiamide-treated group. Gd-DTPA-BMA 1 Fe-Tf % Gd-Tf 1 Fe-DTPA-BMA
Important macroscopic and pathologic lesions were observed There are 2 binding sites for Fe31 with Tf (log Kcond(1) 5
in the nonformulated gadodiamide group. These findings are con- 20.2 and log Kcond(2) 5 39.3).42 A transmetallation phenomenon
sistent with previously reported studies.18,24 involving one site of iron binding on Tf therefore seems possible
Treatments did not modify the CD34 or TGFb1 expression in (firstly dissociation of Gd-DTPA-BMA followed by transligation of
either of the 2 studies (ie, sacrifice at day 11 or 32). In the present iron between 1 binding site of Tf and DTPA-BMA). However, an
work, the expression of these markers was investigated in the increase in plasma levels of iron in healthy volunteers was reported
papillary dermis. The only study in which GC were administered in in the phase I study of gadopentetate.43 The subsequent optimization
rats with impaired renal function under conditions strictly similar to of the gadopentetate pharmaceutical formulation with the addition of
those of the present protocol did not provide histology results.14 In 0.1% of the free ligand DTPA restored normal plasma iron levels in
one study,24 positive CD34 expression was observed in the dermis healthy volunteers.43 Therefore, our data are not consistent with
5 days after the last of 20 injections in rats treated with nonformu- these results.
lated gadodiamide, formulated gadodiamide, and low stability Gd- Another hypothesis may be proposed: typically, an inflam-
EDTA. No CD34 expression was observed in saline-treated rats, in matory process is associated with anemia and a decrease in serum
contrast with our study. This discrepancy might be explained by iron levels.44 Although systemic inflammation was clearly found in
differences in the origin of the anti-rat CD34 antibodies used. In nonformulated gadodiamide-treated rats, no obvious anemia was
another study,18 no expression of CD34 was observed 1 day after the observed. The killing of our rats might have been too early to
13th dose of 5 or 10 mmol/kg gadodiamide or gadopentetate in rats observe anemia.
with normal renal function or 3 days after the 10th dose in SNx rats. It has been proposed that the FGF-23 pathway may be
However, no detailed data were provided with regard to CD34 involved in the pathogenesis of NSF.45 FGF-23 is a peptide pre-
staining in the saline-treated rats (the anti-rat CD34 antibody was dominantly expressed by osteocytes (but also by thymus, brain, and
similar to that used in our study). In our study, a potential effect of lymph nodes) that regulates phosphorus homeostasis and vitamin D
GC on CD34 expression was difficult to demonstrate because of the metabolism. FGF-23 inhibits renal and intestinal absorption of
relatively high basal CD34 staining observed in control rats. phosphate by inhibition of Na-Pi type II transporters.46 It has been
Following inflammatory insults, the effect of TGFb was proposed that, in the context of renal insufficiency, impaired expres-
characterized by increased production of ECM components, as well sion of the Klotho protein which is required for FGF-23-mediated
as mesenchymal cell proliferation, migration, and accumulation.34 receptor activation, would lead to hyperphosphatemia, and via
Tissue fibrosis is primarily attributed to the TGFb1 isoform,35 which negative feedback, to further overexpression of FGF-23 that may
was investigated in our study. TGFb1 plays an important profibrotic eventually lead to fibroblast proliferation.45 In the present study,
role by inducing the synthesis of a-smooth muscle actin and colla- plasma FGF-23 levels were significantly increased in the commer-
gen.35,36 Elevated tissue levels of TGFb mRNA have been identified cial gadodiamide-treated group versus controls and gadoterate-
in NSF.37 However, to our knowledge, this marker has never been treated groups. Interestingly, plasma phosphorus levels were signif-
investigated to date in preclinical, in vivo studies. In the present icantly reduced in the gadodiamide-treated group versus control and
study, relatively high TGFb1 expression was observed in saline- gadoterate groups. It may be speculated that this effect is the

consequence of the hypophosphatemic effects of FGF-23. Subtotal patients with chronic kidney disease who received gadopentetate dimeglu-
nephrectomy did not affect plasma phosphorus levels. This is con- mine. Invest Radiol. 2009;44:135–139.
sistent with the published data.47 It would be of interest to investi- 10. Idée JM, Port M, Medina C, et al. Possible involvement of gadolinium
chelates in the pathophysiology of nephrogenic systemic fibrosis: a critical
gate the effects of GC in SNx rats receiving a phosphate-enriched review. Toxicology. 2008;248:77– 88.
diet to achieve hyperphosphatemia. 11. Morcos SK. Nephrogenic systemic fibrosis following the administration of
The mechanism of NSF remains speculative, and the exact role extracellular gadolinium-based contrast agents: is the stability of the contrast
of GC is still unclear. Many authors agree that the less stable, linear, and agent molecule an important factor in the pathogenesis of this condition? Br J
nonionic GC confer a higher risk for NSF.5,48 –50 This has led to Radiol. 2007;80:73–76.
speculations concerning gradual GC dissociation in the body, associ- 12. Perazella MA. Tissue deposition of gadolinium and development of NSF: a
ated with a subsequent proinflammatory cascade of events leading to convergence of factors. Semin Dial. 2008;21:150 –154.
aberrant systemic fibrosis.11,12 The inverse correlation between total 13. Newton B, Jiménez SA. Mechanism of NSF: new evidence challenging the
prevailing theory. J Magn Reson Imaging. 2009;30:1277–1283.
gadolinium levels in the skin of naive or renally impaired rats and the
14. Pietsch H, Lengsfeld P, Steger-Hartmann T, et al. Impact of renal impairment
stability of administered GC as well as the occurrence of NSF-like skin on long-term retention of gadolinium in the rodent skin following the
lesions in rats exclusively receiving less stable GC,24,51 are strongly administration of gadolinium-based contrast agents. Invest Radiol. 2009;44:
suggestive of a causal role for dissociated Gd31. However, clinical data 226 –233.
indicate that gadolinium is necessary but not sufficient to trigger NSF.10 15. Mahl A, Heining P, Ulrich P, et al. Comparison of clinical pathology
The role of cofactors, found in case-control studies (hyperphos- parameters with two different blood sampling techniques in rats: retrobulbar
phatemia, iron, erythropoietin, renal failure, etc), is indeed a major issue plexus versus sublingual vein. Lab Anim. 2000;34:351–361.
and should be investigated in preclinical studies. Cofactors can be used 16. Crissman JW, Goodman DG, Hildebrandt PK, et al. Best practices guideline:
toxicologic histopathology. Toxicol Pathol. 2004;32:126 –131.
to increase the sensitivity of in vivo models, as recently shown with
17. Milliken GA. Analysis of repeated measures design. In: Berry DA, ed.
erythropoietin and iron.52 Statistical Methodology in the Pharmaceutical Sciences. New York, NY:
In summary, after intravenous administration in rats with im- Dekker; 1990:83–116.
paired renal function, neither gadoterate nor gadodiamide induced 18. Grant D, Johnsen H, Juelsrud A, et al. Effects of gadolinium contrast agents
macroscopic skin lesions, in contrast with nonformulated gadodiamide, in naïve and nephrectomised rats: relevance to nephrogenic systemic fibrosis.
which was associated with a high systemic toxicity. Histopathologic Acta Radiol. 2009;50:156 –169.
lesions were in the range nonformulated gadodiamide . gadodiamide 19. Haylor J, Dencausse A, Vickers M, et al. Nephrogenic gadolinium distribu-
. gadoterate (limited dermal changes). In the long-term study, small tion and skin cellularity following a single injection of gadodiamide in the rat.
Invest Radiol. 2010;45:507–512.
clusters of damaged collagen bundles were found in 3 of 4 gadodia-
20. Ersoy H, Rybicki FJ. Biochemical safety profiles of gadolinium-based extra-
mide-treated rats. Significantly higher plasma FGF-23 levels (associ- cellular contrast agents and nephrogenic systemic fibrosis. J Magn Reson
ated with a decrease in phosphate levels) were measured in the ga- Imaging. 2007;26:1190 –1197.
dodiamide treated-group than in the saline and gadoterate groups. 21. US Food and Drugs Administration CfDEaR. Guidance for Industry.
Decreased plasma transferrin-bound iron levels were measured in the Estimating the maximum safe starting dose in initial clinical trials for
nonformulated gadodiamide group. Overall, the high stability gadoter- therapeutics in adult healthy volunteers; 2005. Available at: www.fda.gov/
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22. Bousquet JC, Saini S, Stark DD, et al. Gd-DOTA: characterization of a new
Further studies are needed to better understand the interaction of paramagnetic complex. Radiology. 1988;166:693– 698.
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ACKNOWLEDGMENTS 24. Sieber MA, Pietsch H, Walter J, et al. A preclinical study to investigate the
The authors thank Dr. Anthony Saul and Dr. Dominique development of nephrogenic systemic fibrosis: a possible role for gadolinium-
Debize-Henderson for reviewing the English version of the based contrast media. Invest Radiol. 2008;43:65–75.
manuscript. 25. Harpur ES, Worah D, Hals PA, et al. Preclinical safety assessment and
pharmacokinetics of gadodiamide injection, a new magnetic resonance im-
aging contrast agent. Invest Radiol. 1993;28:S28 –S43.
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British Journal of DOI:10.1111/j.1476-5381.2011.01585.x
BJP Pharmacology
www.brjpharmacol.org
RESEARCH PAPER bph_1585 1151..1162

Correspondence
Nathalie Fretellier, Guerbet,
Research Division, BP 57400,
Hyperphosphataemia Roissy Charles de Gaulle Cedex,

France. E-mail:
nathalie.fretellier@
sensitizes renally impaired guerbet-group.com

----------------------------------------------------------------
Keywords
rats to the profibrotic nephrogenic systemic fibrosis;
hyperphosphataemia; renal
impairment; gadolinium chelates;
effects of gadodiamide relaxometry; gadolinium;

magnetic resonance imaging;
contrast agents
----------------------------------------------------------------
N Fretellier1,5, JM Idée1, P Bruneval2,7, S Guerret3, F Daubiné4, G Jestin1, Received
C Factor1, N Poveda1, A Dencausse1, F Massicot5, O Laprévote5,6, 1 April 2011
C Mandet7, N Bouzian1, M Port1 and C Corot1 Revised
20 June 2011
1
Guerbet, Research Division, Aulnay-sous-Bois, France, 2Department of Pathology, Hôpital Accepted
Européen Georges Pompidou, Paris, France, 3Novotec, Lyon, France, 4Atlantic Bone Screen, 27 June 2011
Nantes, France, 5Chimie Toxicologie Analytique et Cellulaire, EA 4463, Faculté des Sciences
Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France, 6Service de Toxicologie
Biologique, Hôpital Lariboisière, Paris, France, and 7INSERM U970, Department of Pathology,
Hôpital Européen Georges Pompidou, Paris, France
BACKGROUND AND PURPOSE

Hyperphosphataemia is common in patients with nephrogenic systemic fibrosis (NSF). NSF has been linked to administration
of gadolinium (Gd) chelates (GCs) and elevated serum phosphate levels accelerate the release of Gd from linear, non-ionic
GCs but not macrocyclic GCs. Hence, we determined whether hyperphosphataemia is a cofactor or risk factor for NSF by
investigating the role of hyperphosphataemia in renally impaired rats.
EXPERIMENTAL APPROACH
Firstly, the clinical, pathological and bioanalytical consequences of hyperphosphataemia were investigated in
subtotal nephrectomized (SNx) Wistar rats following i.v. administration of the non-ionic, linear GC gadodiamide (5 ¥
2.5 mmol·kg-1·day-1). Secondly, the effects of several GCs were compared in these high-phosphate diet fed rats. Total Gd
concentration in skin, femur and plasma was measured by inductively coupled plasma mass spectrometry (ICP-MS) and free
Gd3+ in plasma by liquid chromatography coupled to ICP-MS. Relaxometry was used to measure dissociated Gd in skin and
bone.
KEY RESULTS
Four out of seven SNx rats fed a high-phosphate diet administered gadodiamide developed macroscopic and microscopic
(fibrotic and inflammatory) skin lesions, whereas no skin lesions were observed in SNx rats treated with saline, the other GCs
and free ligands or in the normal diet, gadodiamide-treated group. Unlike the other molecules, gadodiamide gradually
increased the r1 relaxivity value, consistent with its in vivo dissociation and release of soluble Gd.
CONCLUSIONS AND IMPLICATIONS

Hyperphosphataemia sensitizes renally impaired rats to the profibrotic effects of gadodiamide. Unlike the other GCs
investigated, gadodiamide gradually dissociates in vivo. Our data confirm that hyperphosphataemia is a risk factor for NSF.
Abbreviations
a-SMA, a-smooth muscle actin; DTPA, diethylene triamine pentaacetic acid; EC, endothelial cells; ECM, extracellular
matrix; Gd, gadolinium; GC, gadolinium chelates; HPLC, high performance liquid chromatography; ICP-MS, inductively
coupled plasma mass spectrometry; MRI, magnetic resonance imaging; NSF, nephrogenic systemic fibrosis; P-4-OH,
prolyl-4-hydroxylase; SNx, subtotal nephrectomy; TIMP-1, tissue inhibitor of metalloproteinase-1; DOTA,
1,4,7,10-tetraazacyclododecane-N,N′,N′,N″-tetraacetic acid
© 2011 The Authors British Journal of Pharmacology (2012) 165 1151–1162 1151
British Journal of Pharmacology © 2011 The British Pharmacological Society
N Fretellier et al.
BJP
Introduction the other categories of GCs and the free ligands Ca-DOTA and
Ca-DTPA. In gadodiamide-treated rats, increased r1 relaxivity
Nephrogenic systemic fibrosis (NSF) is a rare and highly values were found in both femur and skin. These results
debilitating systemic fibrosing disorder occurring in patients indicate gradual in vivo dissociation of gadodiamide, whereas
with severe or end stage renal failure, usually those requiring the other GCs remained stable.
dialysis (Cowper et al., 2001). NSF is typically characterized
by extensive thickening and hardening of the skin associated
with contractures around the joints impairing mobility, pru- Methods
ritus and sharp pain. Histologically, NSF is characterized by
dermal fibrosis with CD34 and procollagen 1-positive cells,
prominent collagen bundles, frequent presence of myxoid
Animals
All studies were carried out on male Wistar rats from Centre
substance between fibroblasts and collagen bundles, and
d’Elevage René Janvier (CERJ) (Le Genest Saint-Isle, France),
increased numbers of macrophages and factor XIIIa-positive
aged 6 weeks and weighing 170 6 7 g. The animals under-
dendritic cells (Braverman and Cowper, 2010).
went one-step subtotal (5/6) nephrectomy performed at
The link between administration of gadolinium (Gd) che-
CERJ: animals were anaesthetized with ketamine
lates (GCs), used as contrast agents for magnetic resonance
(100 mg·kg-1) and xylazine (10 mg·kg-1). The right kidney was
imaging (MRI), and the disease, suggested for the first time in
exposed via a flank incision, the adrenal gland was separated
2006 (Grobner, 2006), is now acknowledged. Briefly, there are
from the upper pole and the kidney was decapsulated. The
two structurally distinct categories of GCs: (i) ‘macrocyclic’
renal pedicle was ligated and the right kidney was removed.
molecules in which the Gd3+ ion is ‘caged’ into the pre-
The left kidney was subsequently decapsulated and the
organized cavity of the ligand; and (ii) ‘linear’ chelates in
adrenal gland was separated from the upper pole. Ligatures
which the ligand is wrapped around the metal ion. GCs can
were placed around the upper and lower poles and the poles
also be either ‘non-ionic’ (in which case three carboxylate
were then excised. An intravenous injection of 1.0 mL·kg-1 of
functions neutralize the three positive charges of Gd3+) or
saline was performed at completion of surgery to compensate
‘ionic’ (where additional carboxylic acid groups are salified
for blood loss. The animals were housed one per cage at an
with either meglumine or sodium) (Port et al., 2008). GCs
ambient temperature of 22 6 2°C, hygrometry of 45 6 10%,
differ in terms of their thermodynamic and kinetic stabilities.
with 12/12 light/dark cycles. Animals had free access to water
High kinetic stability provided by the macrocyclic structure
and animal chow.
combined with high thermodynamic stability minimize the
All animal care and experimental procedures were carried
amount of free Gd3+ that can be released from the parent
out according to French regulations and in compliance with
chelate (Port et al., 2008). Virtually all published cases of NSF
the European Economic Community Directive (2010/63/EU)
have been associated with prior administration of linear GCs
on animal welfare. The protocol was approved by the local
(Broome, 2008; Perazella and Reilly, 2011).
ethics committee.
The mechanism of NSF is not fully understood despite
extensive research (Vakil et al., 2009; Varani et al., 2009;
Edward et al., 2010). Clinically relevant preclinical models are Role of hyperphosphataemia (Study 1)
needed to more clearly understand the mechanism of this Male Wistar rats subjected to subtotal (5/6) nephrectomy (n =
disease. Rats submitted to subtotal (5/6) nephrectomy (SNx) 7 to 8/group) received a normal phosphate (0.6%) diet or a
have been widely used as a model of NSF (Grant et al., 2009; high-phosphate (1.1%) diet and were allocated to single daily
Pietsch et al., 2009; Haylor et al., 2010; Fretellier et al., 2011a). i.v. injections of 2.5 mmol·kg-1 (5.0 mL·kg-1) of gadodiamide
Overall, these studies concluded that fibrotic-like lesions can or saline (5.0 mL·kg-1) for 5 consecutive days, starting 10 days
be induced in rats treated with the linear GC gadodiamide. after subtotal nephrectomy. The 1.1% phosphate diet was
Elevated plasma phosphate levels have been shown to given to rats throughout their lifespan, including in utero
accelerate the release of free Gd3+ from linear, non-ionic GCs period as their mothers were fed with this diet from the 11th
such as gadodiamide, whereas macrocyclic GCs remained day of gestation. For Gd measurement, a skin biopsy was
stable in human serum at both normal and high phosphate performed on Day 4 after the first administration, with a
levels (Frenzel et al., 2008). As higher serum phosphate levels biopsy punch (6 mm diameter) and the wounds were then
have been reported in patients with nephrogenic systemic sutured. The rats were killed (exsanguination under isoflu-
fibrosis compared with sex- and age-matched renal failure rane anaesthesia) 11 days after the first administration. All
control patients (Marckmann et al., 2007), the purpose of the injections and skin samples were performed under isoflurane/
present study was to: (i) investigate the clinical and patho- oxygen (3.5% induction, then 2.5%, 1 L·min-1 oxygen)
logical consequences of hyperphosphataemia in renally anaesthesia.
impaired rats following administration of the linear, non-
ionic GC gadodiamide; and (ii) compare the clinical and Comparison of various GCs and ligands
biochemical effects of all categories of GCs and free ligands in SNx and rats fed a high-phosphate
on SNx rats fed a high-phosphate diet and the possibility of in diet (Study 2)
vivo dissociation of GCs, by using the relaxometry method Male Wistar rats subjected to subtotal (5/6) nephrectomy (n =
(Fretellier et al., 2011b). 6 to 8/group) received a high-phosphate (1.1%) diet and were
Our data indicate that hyperphosphataemia sensitizes allocated to single daily i.v. injections of 2.5 mmol·kg-1 of
renally impaired rats to the profibrotic effects of the linear gadoterate, gadodiamide, gadobenate, gadobutrol or
and non-ionic GC gadodiamide. No effects were observed for 0.5 mmol·kg-1 of the ligands Ca-DTPA or Ca-DOTA or saline
1152 British Journal of Pharmacology (2012) 165 1151–1162

Rat model of nephrogenic systemic fibrosis
BJP
used as control (5.0 mL·kg-1) for 5 consecutive days, starting taken from the sublingual vein (Mahl et al., 2000). All assays
10 days after subtotal nephrectomy. Skin biopsies were per- were performed in duplicate.
formed on Days 1 and 8 after the first administration. The rats
were killed 25 days after the first administration. Gadolinium determination
In Study 2, Gd levels in plasma, skin, liver and femoral epi-
physeal samples, were measured by inductively coupled
Macroscopic skin findings and histopathology plasma mass spectrometry (ICP-MS) on ELAN DRC Plus®
All rats were examined for macroscopic skin changes before (PerkinElmer Life and Analytical Sciences, Inc., Waltham, MA,
the first injection and then daily throughout the study. Skin USA). A standard curve of inorganic Gd (0.64 nmol·L-1 to
biopsy samples were fixed in 4% neutral buffered formalin. 1.30 mmol·L-1) in 6.5% HNO3 was used by monitoring the
After routine dehydration, the samples were embedded in signal of the 157Gd isotope. The acceptance limits (total error)
paraffin, sectioned (5 mm thickness) and stained by were set at 614%. Results are expressed in nmol Gd·g-1 wet
haematoxylin-eosin-saffron staining for histopathological tissue weight (tissues samples) or mmol·L-1 of plasma. The limit
analysis (Studies 1 and 2), picrosirius red to detect the pres- of quantification was 0.64 nmol·L-1. Samples above the limit
ence of collagen (Study 1) and Alcian blue for acidic muco- of detection (0.19 nmol·L-1) but below the limit of quantifi-
polysaccharides (Study 1). cation were given an arbitrary value of 0.32 nmol·L-1. The
Immunohistochemical staining was performed in both dissociated Gd3+ concentration was measured by high perfor-
studies to detect CD34 and TGFb1 using anti-CD34 goat anti- mance liquid chromatography (HPLC) connected to an
body (diluted to 1:1000, R&D Systems, Minneapolis, MN, ICP-MS system as described elsewhere (Fretellier et al., 2011b).
USA) and anti-TGFb1 rabbit antibody (diluted to 1:250,
GeneTex, San Antonio, TX, USA) using ImmPRESS polymer-
ized reporter peroxidase staining (ImmPRESS anti-goat Ig Relaxometry measurements
[peroxidase] kit [MP 7405] and ImmPRESS anti-rabbit Ig [per- In Study 2, the presence of dissociated Gd in skin biopsies and
oxidase] kit [MP 7401], respectively; Vector Laboratories Bur- trabecular femur was assessed by relaxometry technique
lingame, CA. USA). because mass spectrometry cannot characterize the exact
In Study 2, in addition to CD34 and TGFb1, immun- nature of Gd species in solid tissues. This technique has been
ostaining of S100A4 (i.e. fibroblast-specific protein-1) (rabbit described elsewhere (Fretellier et al., 2011b). Briefly, the
antibody diluted to 1:500, Abcam, Paris, France), using anti- samples were mashed, differently according to the matrix,
rabbit biotinylated antibody (AbCys, Paris, France) staining, and were diluted (1/11) in D2O/H2O (90/10) mix. Longitudi-
a-smooth muscle actin (a-SMA) (mouse antibody diluted to nal relaxation times (T1) were measured on Bruker Minispec
1:600, Thermo Fisher Scientific, Brebières, France) using anti- (Bruker Optics, Ettlingen, Germany) at 60 MHz (i.e. 1.42 T)
mouse biotinylated antibody (AbCys) staining, macrophages and 37°C.
(ED-1 stain) (mouse antibody diluted to 1:100, AbD Serotec, The r1 relaxivity value could not be determined when the
Colmar, France) using anti-mouse biotinylated antibody 1/T1sample – 1/T1diamagnetic value was less than 20% of
(AbCys) staining, tissue inhibitor of metalloproteinase-1 1/T1diamagnetic in the absence of Gd precipitation (i.e. because of
(TIMP-1) (goat antibody diluted to 1:100, R&D Systems, Min- a low total Gd concentration in the sample). The Gd concen-
neapolis, MN, USA) using anti-goat biotinylated antibody tration was then determined by ICP-MS. The Gd concentra-
(AbCys) staining and prolyl-4-hydroxylase (P-4-OH) (mouse tion in tissue samples was corrected for an estimated 97%
antibody diluted to 1:100, Acris Antibodies GmbH, Herford, water content, based on unpublished in-house studies. Relax-
Germany) using anti-mouse biotinylated antibody (AbCys) ivities (r1 apparent value) were calculated according to the
staining, was performed at the end of the study. Histopatho- formula: r1 = (1/T1sample – 1/T1diamagnetic)/[Gd], where relaxation
logical lesions were evaluated semi-qualitatively using a rate (1/T1) was expressed in s-1, Gd concentration in mM and
4-point severity grading scale (- absent; 6 mild; + moderate; relaxivity r1 in mM-1·s-1.
++ severe) for each animal. Histopathological examinations Relaxometry studies (in vitro spiking studies) on biological
were performed in a blinded fashion by certified histo- matrices were performed by spiking with GC (from commer-
pathologists (S. G. in Study 1 and P. B. in Study 2) in accor- cial solutions) (range of 0, 0.005, 0.01, 0.02, 0.04, 0.05, 0.1,
dance with the guidelines of the Society of Toxicological 0.5 and 1 mM) on D2O/H2O mix, rat plasma, skin and trabe-
Pathology (Crissman et al., 2004). cular femur from untreated rats. The term ‘in vitro studies’
refers to all experiments performed by spiking GC on tissue
matrices (to obtain the reference range of r1 relaxivities) and
Biochemistry ‘in vivo studies’ refers to all experiments performed on test
In Study 2, the plasma levels of total calcium, phosphorus, animals. Based on unpublished in vitro relaxometry studies,
transferrin-bound iron and creatinine (Vitros-II autoanalyzer, the uncertainty of relaxivity r1 measurements (with Gd con-
OrthoClinical Diagnostics Inc., Issy-les-Moulineaux, France) centration measured by ICP-MS) was set at r1 623%. The in
were measured on Days 0 (i.e. 3 days before the first admin- vitro r1 relaxivity range represents the in vitro r1 relaxivity
istration), 5 and 25. In addition, plasma levels of monocytes value (obtained from spiking studies) 6 uncertainty of 23%.
chemotactic protein-1 (MCP-1) (Bender MedSystems, Vienna,
Austria), TIMP-1 and TGFb1 (R&D Systems, Minneapolis, MN, Statistical analysis
USA) were measured by ELISA on Day 5. Plasma levels of TGFb1 Data are expressed as mean 6 SD. Statistical analysis was
and fibroblast growth factor-23 (Kainos Laboratories, Tokyo, carried out using GraphPad Prism 4 (GraphPad Software Inc,
Japan) were measured by ELISA on Day 25. Blood samples were San Diego, CA, USA). Results were analysed by repeated-
British Journal of Pharmacology (2012) 165 1151–1162 1153

N Fretellier et al.
BJP
measures ANOVA (body weight, biochemistry data, plasma Gd Comparison of various GCs and ligands in
concentration) or by one-way ANOVA (Gd concentrations) rats fed a high-phosphate diet (Study 2)
followed, when relevant, by a Bonferroni or Dunnett’s test. Macroscopic and microscopic findings. Ulcerative and squa-
Skin relaxivity values were analysed by Student’s t-test. Dif- mous skin eruptions occurred (from Day 8 until Day 23) in
ferences were considered significant when P < 0.05. five of the eight rats receiving gadodiamide, and worsened in
four animals or improved in one, while no skin lesions were
observed in the rats treated with saline, the other GCs and
Materials the free ligands. Four gadodiamide-treated rats had to be
Meglumine gadoterate (Dotarem, batch 09GD022A, Guerbet, killed for ethical reasons because of the severity of the skin
Villepinte, France), gadodiamide (Omniscan, batch 10758384 lesions and loss of body weight (one rat was killed at Day 5,
and batch 10942539, General Electrics Healthcare, Chalfont one at Day 15, one at Day 22 and one at Day 23). No scratch-
St Giles, UK), dimeglumine gadobenate (MultiHance, batch ing was observed. One gadoterate-treated rat was found dead
S9P252A, Bracco, Milan, Italy), gadobutrol (Gadovist, batch at Day 8. No significant differences in body weight changes
9A033A, Bayer Healthcare, Berlin, Germany) were purchased
were observed between these groups.
from respective manufacturers. The ligands Ca-DTPA (dieth-
On histological examination, the skin was considered to
ylene triamine pentaacetic acid) and Ca-DOTA (1,4,7,
be abnormal in six of the eight rats of the gadodiamide-
10-tetraazacyclododecane-N,N′,N′,N″-tetraacetic acid) were
treated group (three rats with macroscopic skin lesions
synthesized at Guerbet Research Dpt. Saline was purchased
and killed, one rat without macroscopic skin lesions and
from Lavoisier (Paris, France). killed and two rats without skin lesions and not killed
before the end of the experiment), while no histological
skin changes were observed in the other groups. Lesions
consisted of bands of inflammatory dermal fibrosis associ-
Results ated with hyperkeratosis and calcifications (Figure 2). ED-1,
TIMP-1 and TGFb1-positive macrophages accumulated in
Characterization of the experimental model the dermis (Figure 2, Table 1) and were associated with an
An increase in plasma creatinine (66.8 6 20.1 vs. 25.8 6 increased density of CD34+ and S100A4+ spindle cells.
2.1 mmol·L-1, P < 0.01) and phosphorus (3.4 6 0.8 vs. 2.0 6 Positive immunostaining for TGFb1, TIMP-1 and P-4-OH
0.1 mmol·L-1, P < 0.01) levels was observed in SNx rats fed the (Figure 2) was observed for spindle cells of gadodiamide-
high-phosphate diet compared with sham operated rats. treated rats (6/8). No a-smooth muscle actin expression
Plasma calcium levels remained unchanged (2.1 6 0.2 vs. 2.4 was detected, irrespective of the group. In all other groups
6 0.1 mmol·L-1, not significant). Pathological lesions consis- and in the controls, immunohistochemical patterns were
tent with osteodystrophy (osteitis fibrosa) were found in both normal except for one rat treated with gadobenate, in
the femur and tibia of SNx rats fed a high-phosphate diet which a fibro-inflammatory focus located in the dermis
(data not shown). with the presence of ED-1 and TGFb1-positive cells was
observed.
Role of hyperphosphataemia (Study 1)

Macroscopic and microscopic findings. The plasma phosphate Biochemistry. No significant differences between the groups
level was higher in SNx rats receiving a high-phosphate diet were found regarding the plasma levels of all tested param-
than in SNx rats fed a normal diet (2.4 6 0.4 and 1.9 6 eters at Day 0. The plasma creatinine level of all treated-
0.2 mmol·L-1, respectively; P < 0.05). Plasma calcium levels groups was consistent with renal impairment (data not
were similar in the two groups (2.2 6 0.2 and 2.3 6 shown). A slight and non-significant decrease in plasma
0.2 mmol·L-1, respectively; not significant). No skin lesions phosphorus levels was observed from Day 5 in the gadodia-
were observed in the gadodiamide- and saline-treated groups mide, gadobutrol, gadobenate and Ca-DOTA-treated groups
receiving a normal-phosphate diet, while four out of the (Table 2). A substantial decrease in plasma phosphorus levels
seven gadodiamide-treated rats fed a high-phosphate diet (-30%) was observed at Day 5 in the Ca-DTPA-treated group
showed macroscopic skin lesions (ulcerations and scabs) in (P < 0.05 vs. Day 0 values).
the neck, back and abdominal skin for the first time on Day A slight and non-significant increase in plasma iron con-
8 (i.e. after the first injection). No significant differences in centration was also observed on Day 5 in saline, gadoterate,
body weight changes were observed between these groups. gadobutrol and Ca-DOTA-treated groups. Because analytical
Histologically, small superficial epidermal lesions, degra- interference of iron measurement was observed with
dation and merging of collagen fibres in the dermis with Ca-DTPA, gadobenate and gadodiamide, no results are pro-
disorganization of the extracellular matrix (ECM) were vided for these compounds (Table 2).
observed in the gadodiamide group fed with normal diet, No significant differences in plasma levels of TGFb1 and
while numerous zones of epidermal necrosis, a moderately fibroblast growth factor-23 were observed between the
increased dermal cellularity, the presence of multinucleated various groups (data not shown). The GCs and ligands had no
giant cells, more abnormalities of dermal collagen structures effect on plasma levels of MCP-1 and TIMP-1 (measured on
(dense and short collagen bundles alternating with altered Day 5) (data not shown). However, an increase in the plasma
collagen fibres) and more intense TGFb1 immunostaining levels of MCP-1 (17 times vs. controls) and TIMP-1 (seven
were observed in the gadodiamide-treated rats fed a high- times vs. controls) was observed in one gadodiamide-treated
phosphate diet (Figure 1). rat (this animal was killed at Day 5).

BJP
Gadodiamide and high-
Saline Gadodiamide phosphate diet
HES
A B C
Picrosirius red
D E F
TGFβ1
G H I
Figure 1
Histological skin lesions in the papillary dermis: haematoxylin and eosin staining (A–C) to analyse cellular lesions, picrosirius red staining (D–F) to
detect changes in extracellular matrix and TGFb1 (G–I) to detect spindle cells (Study 1). Saline (A, D, G): epidermal thickness was constant; the
dermis was weakly cellular, and ECM was homogeneous. TGFb1 was only expressed on several spindle cells. Gadodiamide (B, E, H): local epidermal
necrosis and presence of some inflammatory cells in the papillary (or superficial) dermis. ECM was heterogeneous, with small clusters of collagen
fibres. TGFb1 immunohistochemical detection was slightly increased in papillary and reticular dermis. Gadodiamide in SNx rats fed a high-
phosphate diet (C, F, I): numerous foci of epidermal necrosis; some foci of myxoid tissue, necrosis, presence of multinucleated giant cells, [Gc]
included in fragmented collagen fibres, were observed (C, F), as well as an increase in subepithelial TGFb1 immunostaining (I). (F) Shows short
and dense collagen bundles alternating with altered fibres. D, dermis; E, epidermis; HES, haematoxylin-eosin-saffron.
Plasma levels of total and dissociated gadolinium. No signifi- total Gd concentration at Day 25 (vs. Day 8) (Figure 4) was
cant differences in plasma total Gd levels were observed in observed in the gadoterate, gadobutrol and gadobenate-
the gadoterate, gadodiamide or gadobutrol-treated groups treated groups. The total Gd concentration in dorsal skin was
(Figure 3), while plasma total Gd levels were lower with gado- higher (P < 0.001) with gadodiamide (153.0 6 82.7 nmol·g-1)
benate than with the other GCs (P < 0.05 at Day 8). No than in the other GC-treated groups at Day 25.
dissociated Gd3+ was found in the plasma of gadobutrol and
gadobenate-treated rats, regardless of the time-point, while Total gadolinium levels in the femur and liver. Total Gd con-
two rats (out of eight) showed the presence of Gd3+ levels at centrations in the femur and liver were higher in the
Day 8 in the gadoterate treated-group (Table 3). Marked gadodiamide-treated group than in the other treated groups
release of Gd3+ was observed with gadodiamide in all treated (P < 0.001) (Figure 5A and B).
animals (Table 3).
Relaxometry studies. The r1 relaxivity values obtained in
Total gadolinium levels in skin. No significant differences in water (90/10 D2O/H2O solution), rat skin, femur, plasma and
total Gd concentration in skin at Day 8 were observed liver matrices (in vitro ‘spiking’ studies) are shown in Table 4.
between treated groups except for gadobenate-treated rats, in In in vivo studies, the r1 relaxivity values of gadoterate or
which the Gd concentration was significantly lower than gadobutrol were situated in the r1 in vitro range in skin (Days
in the other groups (Figure 4). A dramatic decrease in the skin 1 and 8) and femur (Figures 6 and 7). However, in 19/25 rats

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Figure 2
Histological (haematoxylin-eosin-saffron [HES]), and immunohistochemical (ED-1, TGFb1, prolyl-4-hydroxylase, TIMP-1) analysis of dorsal skin in
SNx rats fed a high-phosphate diet (typical examples from Study 2). Bands of inflammatory dermal fibrosis associated with hyperkeratosis and
calcifications were observed (HES) as well as the presence of ED-1 + macrophages, TGFb1, prolyl-4-hydroxylase and TIMP-1 immunostaining in
the dermis.
Table 1
Semi-quantitative immunohistochemical analysis of dorsal skin (number of rats with positive immunostaining)
No of
Treatment rats CD34 ED-1 TGFb1 S100A4 a-SMA P-4-OH TIMP-1
Saline 5 (+) [sc and ECs] (6) (-) (+) [sc] (-) (6) (6)
Gadoterate 8 (+) [sc and ECs] (6) (-) (+) [sc] (-) (6) (6)
Gadodiamide 8 (+) [sc and ECs] but (6) = 3 (-) = 2 (+) [sc] but ↑ (-) (6) = 2 (6) = 2
↑ number of sc (++) = 5 (foci) = 2 number of sc Foci (++) = 6 Foci (++) = 6
(+) = 1
(++) = 3
Gadobenate 7 (+) [sc and ECs] (6) = 6 (-) = 6 (+) [sc] (-) (6) (6)
(++) = 1 (focus) (++) = 1 (focus)
Gadobutrol 7 (+) [sc and ECs] (6) (-) (+) [sc] (-) (6) (6)
Ca-DOTA 6 (+) [sc and ECs] (6) (N/A) (N/A) (N/A) (N/A) (N/A)
Ca-DTPA 6 (+) [sc and ECs] (6) (N/A) (N/A) (N/A) (N/A) (N/A)
‘sc’ indicates spindle cells and ‘ECs’ indicates endothelial cells. - = absence, 6 = mild, + = moderate, ++ = severe, N/A = not available.
(gadoterate, gadobutrol and gadobenate), the r1 value could relaxivity value (38.2 6 10.4 mM-1·s-1) was observed in the
not be determined at Day 25 because the 1/T1sample – liver in the gadobenate-treated group, compared with the in
1/T1diamagnetic value was less than 20% of 1/T1diamagnetic (low total vitro r1 relaxivity value (4.9 mM-1·s-1).
Gd concentration in the samples). A slight and non-
significant increase in the r1 value was observed at Day 8 (vs.
Day 1 value) in skin samples from gadobenate-treated rats. Discussion and conclusions
A gradual increase in the r1 relaxivity value (P < 0.01 at
Day 8 vs. Day 1 and P < 0.05 at Day 25 vs. Day 1) was Most patients with end-stage renal failure present hyper-
observed in the dorsal skin of gadodiamide-treated rats phosphataemia, which is associated with secondary hyper-
(Figure 6). parathyroidism, osteodystrophy and increased mortality
In the gadodiamide (all rats) and gadobenate (n = 2 mea- (Coladonato, 2005). In two case-control studies, significantly
surable) groups, the r1 value exceeded the r1 in vitro range in higher serum phosphate levels were observed in NSF patients,
femur samples (Figure 7). A dramatic increase in the in vivo r1 compared with patients with chronic kidney disease but with

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Table 2
Plasma phosphorus or iron levels on Day 0 and percentage change (%) of plasma phosphorus or iron levels compared with Day 0
Phosphorus Iron
Plasma (phosphorus) Percentage change Plasma (iron) Percentage change
(mmol·L-1) versus Day 0 (%) (mmol·L-1) versus Day 0 (%)
Treatment Day 0 Day 5 Day 25 Day 0 Day 5 Day 25
Saline 3.3 6 0.8 -5 -10 43.4 6 15.6 +12 -2

Gadoterate 3.0 6 0.2 +6 -1 42.1 6 7.2 +21 +18
§ §
Gadodiamide 3.1 6 0.3 -7 -13 37.1 6 8.3
Gadobutrol 2.7 6 0.4 -8 -15 43.8 6 9.5 +5 +3
§ §
Gadobenate 2.8 6 0.5 -13 -15 47.8 6 9.8
Ca-DOTA 2.8 6 0.3 -8 -15 33.5 6 6.0 +18 +3
§ §
Ca-DTPA 2.8 6 0.2 -30* -13 37.8 6 8.6
*P < 0.05 versus control (saline) group.

§Analytical interference. Uninterpretable data.
Table 3
Dissociated Gd concentration in plasma on Day 8 and Day 15 (HPLC-ICP-MS) and dissociated/total Gd concentration ratio
Day 8 Day 15
Dissociated [Gd3+] in Dissociated/total Gd Dissociated [Gd3+] in Dissociated/total Gd
Treatment plasma (mmol·L-1) concentration ratio (%) plasma (mmol·L-1) concentration ratio (%)
Gadoterate <LOD (n = 6) 36 (n = 2) <LOD (n = 7) –

1.1 (n = 2)
Gadodiamide 2.4 6 0.9 (n = 7) 71 6 23 (n = 7) <LOD (n = 3) 110 6 10 (n = 4)
1.3 6 0.9 (n = 4)
Gadobutrol <LOQ (n = 7) – <LOD (n = 4) –
<LOQ (n = 3)
Gadobenate <LOQ (n = 7) – <LOD (n = 1) –
<LOQ (n = 6)
LOD indicates limit of detection and LOQ indicates limit of quantification.
Figure 4
Total Gd concentration measured in skin samples of rats subjected to
Figure 3 subtotal nephrectomy and high-phosphate diet receiving each GC
Total Gd concentration measured in plasma of rats subjected to (5 ¥ 2.5 mmol·kg-1 IV) on Day 8 and Day 25 (rat killed) (ICP-MS
subtotal nephrectomy and high-phosphate diet receiving each GC measurement). In untreated rats (control group), the total Gd con-
(5 ¥ 2.5 mmol·kg-1 IV) (ICP-MS measurement) between Day 8 and centration in skin was 1.9 6 1.3 nmol·g-1 on Day 8 and 1.7 6
Day 25. *P < 0.05 versus other GCs. 1.4 nmol·g-1 on Day 25. ***P < 0.001 versus all GC treated-groups.

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A B
350 140 ∗∗∗
∗∗∗
Total [Gd3+] in bone at Day 25
300
Total [Gd3+] in liver at Day 25

120
250 100
(nmol·g-1)
(nmol·g-1)
200 80
∗
150 60
100 40
50 20
0 0
Gadoterate Gadodiamide Gadobutrol Gadobenate Gadoterate Gadodiamide Gadobutrol Gadobenate
Figure 5
Total Gd concentration measured in bone (A) or in liver (B) samples of SNx rats fed a high-phosphate diet receiving each GC (5 ¥ 2.5 mmol·kg-1
i.v.) on Day 25 (rat killed) (ICP-MS measurement). In untreated rats (control group), the total Gd concentration was 0.8 6 1.2 nmol·g-1 in bone
samples and below the limit of quantification in liver samples. ***P < 0.001 versus all GC-treated groups. *P < 0.05 versus gadobutrol.
Table 4
In vitro r1 relaxivity value (spiking studies with gadoterate, gadodiamide, gadobutrol or gadobenate [60 MHz, 37°C]) in D2O/H2O and various
tissue matrices from control rats
In vitro r1 relaxivity (mM-1·s-1) (623%)

Matrix Gadoterate Gadodiamide Gadobutrol Gadobenate
D2O/H2O (90/10) 3.3 [2.5–4.1] 3.7 [2.9–4.6] 3.8 [2.9–4.7] 4.8 [3.7–5.9]
Trabecular Femur 3.8 [2.9–4.7] 4.6 [3.5–5.7] 4.5 [3.5–5.6 ] 4.6 [3.5–5.7]
Dorsal skin 3.0 [2.3–3.7] 4.5 [3.5–5.6] 3.5 [2.7–4.3] 4.8 [3.7–5.9]
Plasma 3.7 [2.9–4.6] 4.1 [3.2–5.0] 4.4 [3.4–5.4] 5.7 [4.4–7.0]
Liver – – – 4.9 [3.7–5.9]
The uncertainty of relaxivity r1 measurements (Gd concentration measured by ICP-MS) was set at r1 623%.
Figure 6
Relaxivity r1 values (60 MHz, 37°C) in skin samples of SNx rats fed a high-phosphate diet treated with gadoterate, gadodiamide, gadobutrol or
gadobenate on Days 8 and 25 (rat killed). *P < 0.05 versus gadobenate and gadobutrol. **P < 0.01 versus all GC-treated groups. LOQ indicates
limit of quantification. NS, not significant.

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Figure 7
Relaxivity r1 values (60 MHz, 37°C) in (trabecular) bone samples of SNx rats fed a high-phosphate diet treated with gadoterate, gadodiamide,
gadobutrol or gadobenate on Day 25 (rat killed). LOQ indicates limit of quantification.
no signs of NSF (Marckmann et al., 2007; Prince et al., 2008). scabs). Thus, in addition to impaired renal function, hyper-
The main purpose of our study was to determine whether phosphataemia sensitizes the model. These data are therefore
hyperphosphataemia is a cofactor or a risk factor of NSF as consistent with the role of hyperphosphataemia as a risk
suggested by Peak and Sheller (2007). factor rather than a cofactor in NSF.
The GC administration protocol used in our studies is In the second study, all chemical categories of GCs and
similar to that used by other teams (Grant et al., 2009; Pietsch two polyazapolycarboxylic ligands (calcium complexes of
et al., 2009). Although the dose used was higher than the DOTA and DTPA) were compared on this ‘sensitized’, model
range 0.1 to 0.3 mmol·kg-1 used for MRI contrast examina- of SNx rats fed a high-phosphate diet. Macroscopic (ulcer-
tion in clinical practice, it is appropriate for chronic studies in ations and scabs) and histopathological skin lesions were
the rat because the comparative drug doses between species only observed in gadodiamide-treated animals. The other
should be normalized to body surface area rather than body categories of GCs, which are thermodynamically more stable
weight (US Food and Drugs Administration CfDEaR, 2005). (Port et al., 2008), were not associated with macroscopic or
The SNx and high-phosphate diet rat model used in this microscopic skin lesions. Furthermore, no lesions were found
study is classically used as a model of hyperparathyroidism with the two polyazapolycarboxylic ligands tested, the mac-
(Sanchez et al., 2004; Jiang and Wang, 2008) and bone lesions rocyclic Ca-DOTA and the linear Ca-DTPA. The daily dose of
(Oste et al., 2007). Both the bioavailability of phosphorus and free ligand selected in this study was consistent with the dose
the degree of renal failure are important parameters in the of caldiamide (the free ligand added to the commercial solu-
clinical relevance of the SNx rat model-associated osteodys- tion of gadodiamide) used in another study (Sieber et al.,
trophy (Oste et al., 2007). Phosphorus sources in the standard 2008a).
rat diet present a low bioavailability. However, the high- Bands of dermal inflammatory fibrosis were found in six
phosphate diet used in this study included inorganic Ca/P of the eight gadodiamide-treated rats. Increased cellularity of
sources (monocalcium phosphate) with a high absorption the dermis was also observed. Interestingly, TGFb1 staining
rate, leading to a high bioavailability of phosphorus (Oste was observed on both dermal macrophages and spindle cells.
et al., 2007). Elevated tissue levels of TGFb messenger RNA (mRNA) have
In the first study, histological lesions consistent with been previously identified in NSF (Jiménez et al., 2004). Also
those observed in NSF patients, including a haphazard in another study, an increase in TGFb protein and mRNA
arrangement of short and dense collagen bundles (Cowper levels and Smad-2 and Smad-3 mRNA levels was reported
et al., 2008; Braverman and Cowper, 2010) were observed in (Schieren et al., 2010). Positive immunostaining for TGFb1
SNx rats fed a high-phosphate diet and treated with gadodia- was also demonstrated in NSF skin samples (Kelly et al., 2008)
mide. Multinucleated giant cells were observed, a feature as well as Smad-2 and -3 nuclear staining. These results
sometimes reported in NSF patients (Wilford et al., 2010). suggest an association between TGFb1 and fibrosis in NSF.
Gadodiamide-induced fibrosis-like lesions were more marked TGFb1 is a key mediator in fibrosis, as it induces fibroblasts to
in SNx rats fed a high-phosphate diet than in SNx rats fed synthesize and contract the ECM (LeRoy et al., 1990; Schiller
with normal diet. It has been shown (Fretellier et al., 2011a) et al., 2004). Dermal CD34 expression is an important feature
that no dermal fibrosis was observed up to 32 days after the of NSF (Cowper et al., 2008). It is noteworthy that moderate
first injection of gadodiamide, thus ruling out the possibility CD34 and S100A4 (i.e. fibroblast-specific protein-1) expres-
that high-phosphate diet may have accelerated the response. sion was observed in control biopsy samples. In human skin,
Epidermal lesions were also found in gadodiamide-treated CD34 has been demonstrated in vascular endothelial cells, in
SNx rats fed a high-phosphate diet. However, in rats, micro- a subset of dendritic/spindle-shaped cells (Nickoloff, 1991),
scopic dermal abnormalities should be considered to be more and in some skin tumours (Cohen et al., 1997). The increased
clinically relevant than macroscopic epidermal lesions (e.g. immunostaining of these markers, observed in gadodiamide-

N Fretellier et al.
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treated rats, reflects the increased density of fibroblasts in the effect was found with the macrocyclic GCs gadoterate or
dermis. The CD34+ cells may represent recently migrated gadobutrol. A dramatic increase in the r1 value in liver
CD34+ fibrocytes (bone marrow-derived mesenchymal stem samples was observed in rats treated with gadobenate. A
cells) (Bucala, 2008). Moreover, the possibility of CD34+ possible explanation is that gadobenate binds to albumin.
endothelial cells cannot be ruled out. The r1 relaxivity value at 20 MHz of the free contrast agent
P-4-OH is an essential enzyme in collagen synthesis (without any binding to albumin) (‘r1 free’) is 4.5 mM-1·s-1
(Gorres and Raines, 2010). P-4-OH-positive foci were exclu- and the r1 value of the non-covalently albumin-bound con-
sively found in the dermis of gadodiamide-treated rats. This trast agent (‘r1 bound’) is 36 mM-1·s-1 (Port et al., 2005).
finding is consistent with that of another study (Haylor et al., However, in trabecular femur, the increase in the in vivo r1
2010) in which positive immunohistochemical staining for value was lower than that observed in liver tissue, but higher
this enzyme was found after a single injection of gadodia- than that measured in vitro in bone matrix, suggesting a
mide in renally impaired rats. possible in vivo dissociation of gadobenate in bone tissue. The
Overall, the pathological features observed under these r1 value at Day 25 (in skin and bone) could not be determined
experimental conditions are compatible with those of NSF. in 19/25 rats treated with gadobutrol, gadobenate or gadot-
However, no a-SMA staining was observed in our model, while erate because the 1/T1sample – 1/T1diamagnetic value was less than
a-SMA+ myofibroblasts have been reported in NSF lesions 3 to 20% of 1/T1diamagnetic (low total Gd concentration in samples).
4 weeks old (Swartz et al., 2003; Cowper, 2007). However, it is Hyperphosphataemia probably increases the risk of disso-
noteworthy that in a recent study (Kelly et al., 2010), NSF skin ciation of linear GCs in biological milieu. Frenzel et al. (2008)
samples were negative for a-SMA. The same authors reported showed that Gd3+ release rate for non-ionic linear GCs and
strong expression of TIMP-1 accompanied by almost absent the total amount released after 15 days were increased in
expression of MMP-1. Another study reported increased levels human plasma at 37°C, following addition of 10 mM phos-
of expression of both TIMP-1 mRNA and protein in skin phate, while no effect was observed for macrocyclic GCs. The
samples from NSF patients (Schieren et al., 2010). Under our amount of Gd3+ released from gadobenate was substantially
experimental conditions, TIMP-1 immunostaining was lower than that released from non-ionic linear GCs, but
detected on both dermal macrophages and spindle cells. higher than that released from macrocyclic agents. Alto-
TIMP-1 blocks the activation of MMP-1 (Matrisian, 1990), that gether, these results were consistent with the thermodynamic
plays a pivotal role in collagen degradation. Gadolinium chlo- stability range of GCs (Port et al., 2008).
ride (Bhagavathula et al., 2010) and gadodiamide (DaSilva It may be tempting to link the clinical sensitization of the
et al., 2010) have both been shown to stimulate MMP-1 and SNx model associated with hyperphosphataemia, observed in
TIMP-1 production with no apparent increase in type I pro- Study 1, to phosphate-associated dissociation of gadodiamide
collagen production in cultured human skin fibroblasts. and, subsequently, to a profibrosing effect of dissociated Gd.
Renal dysfunction is the common predisposing factor of However, the protocol used in our study cannot provide any
NSF (Cowper, 2007). Several clinical (Thakral et al., 2007; conclusions concerning this hypothesis, which would require
Abraham et al., 2008) and preclinical studies (Pietsch et al., demonstration of a correlation between skin dissociated Gd
2009 and Pietsch et al., 2011; Sieber et al., 2008a,b) have concentrations and pathological lesions (such as increased
demonstrated that Gd may persist in tissues for long periods cellularity, and/or changes in ECM). However, while relaxom-
of time. In our study, no significant differences in plasma etry can be used to detect dissociated states of Gd in solid
total Gd levels were observed in GC-treated rats, except in tissues such as skin or bone, it cannot quantify this dissocia-
gadobenate-treated rats, in which lower plasma total Gd con- tion. HPLC-ICP-MS allows quantification of dissociated Gd3+
centrations were observed at all time-points. Gadobenate is in plasma but cannot be used in solid tissues, in which it
eliminated in urine and bile (Spinazzi et al., 1999) with would be more pathophysiologically relevant. Therefore, the
intense biliary excretion in rats (50% excretion rate 7 h after bioanalytical tools at the present time are unable to correlate
injection; Lorusso et al., 1999). As the rats used in the present dissociated Gd concentration and skin lesions.
study were renally impaired, it is likely that biliary clearance The question of which Gd species is actually involved in
of gadobenate was increased to compensate for renal impair- the pathogenesis of the lesions observed remains unan-
ment and that total clearance of this compound exceeded swered. Several studies have demonstrated micron-sized
that of the other GCs tested (Ersoy and Rybicki, 2007). insoluble deposits containing Gd associated with calcium and
The Gd concentrations found in the skin, femur and liver phosphate in NSF patients (Thakral and Abraham, 2009;
were higher in gadodiamide-treated rats than in the other George et al., 2010). The protocol used in our study was
GC-treated groups. No difference was observed between the unable to distinguish precipitated insoluble Gd in tissues.
two macrocyclic GCs tested, gadoterate and gadobutrol However, the presence of circulating soluble and probably
(except in the liver). These results are consistent with those of protein-bound Gd was demonstrated in gadodiamide-treated
another study conducted in rats (Sieber et al., 2008b). Pre- rats. All states of Gd (i.e. dissociated and soluble, dissociated
clinical data (Sieber et al., 2008a,b; Pietsch et al., 2009; Steger- and precipitated and/or chelated) may be present in the
Hartmann et al., 2009; Pietsch et al., 2011) are strongly samples. Gadolinium (in the form of GdCl3) has been shown
suggestive of a causal role for dissociated Gd3+. In vivo disso- to promote fibroblast proliferation in vitro (Varani et al., 2009;
ciation of Gd3+ has been found in SNx rats treated with Bhagavathula et al., 2010; Edward et al., 2010), an effect asso-
gadodiamide, while no such effect was observed with the ciated with the formation of insoluble, Gd and phosphorous-
macrocyclic GC gadoterate (Fretellier et al., 2011b). containing aggregated particles (Li et al., 2010).
Our data are consistent with the gradual release of Gd The release of dissociated Gd from a GC must be esti-
from gadodiamide in tissues of treated rats, while no such mated by taking into account both the thermodynamic and

BJP
kinetic stabilities (Idée et al., 2009). It is speculated that, in Cowper SE, Su LD, Bhawan J, Robin HS, LeBoit PE (2001).
the case of low thermodynamic stability but high kinetic Nephrogenic fibrosing dermopathy. Am J Dermatopathol 23:
stability, the timescale would be too long to reach a biologi- 383–393.
cally significant Gd release in animals. Sensitization of NSF Cowper SE, Rabach R, Girardi M (2008). Clinical and histological
models should accelerate Gd dissociation in order to induce findings in nephrogenic systemic fibrosis. Eur J Radiol 66: 191–199.
clinically relevant lesions. In our study, hyperphosphataemia Crissman JW, Goodman DG, Hildebrandt PK, Maronpot RR,
sensitized SNx rats to gadodiamide. This is consistent with Prater DA, Riley JH et al. (2004). Best practices guideline: toxicologic
Frenzel’s data showing a phosphate-related dissociation of histopathology. Toxicol Pathol 32: 126–131.
linear GCs. Nevertheless, in the case of ‘intermediate’ mol-
DaSilva M, Deming MO, Fligiel SE, Dame MK, Johnson KJ,
ecules (i.e. poor thermodynamic stability but high kinetic Swartz RD et al. (2010). Responses of human skin in organ culture
stability, such as gadobutrol), additional sensitization may be and human skin fibroblasts to a gadolinium-based MRI contrast
needed to shorten the timescale in order to evaluate their agent: comparison of skin from patients with end-stage renal
safety under more discriminative conditions. disease and skin from healthy subjects. Invest Radiol 45: 733–739.
In conclusion, hyperphosphataemia sensitizes the effects Edward M, Quinn JA, Burden AD, Newton BB, Jardine AG (2010).
of the linear, non-ionic GC gadodiamide in renally impaired Effect of different classes of gadolinium-based contrast agents on
rats. This GC appears to be the most toxic of the commer- control and nephrogenic systemic fibrosis-derived fibroblast
cially available GCs. Further studies are warranted to better proliferation. Radiology 256: 735–743.
understand the mechanism of NSF and the role of hyperphos-
Ersoy H, Rybicki FJ (2007). Biochemical safety profiles of
phataemia as a risk factor. gadolinium-based extracellular contrast agents and nephrogenic
systemic fibrosis. J Magn Reson Imaging 26: 1190–1197.
Frenzel T, Lengsfeld P, Schirmer H, Hütter J, Weinmann HJ (2008).
Acknowledgements Stability of gadolinium-based magnetic resonance imaging contrast
agents in human serum at 37°C. Invest Radiol 73: 817–828.
The authors thank Drs Anthony Saul and Dominique Fretellier N, Idée JM, Guerret S, Hollenbeck C, Hartmann D,
Debize-Henderson for reviewing the English version of the González W et al. (2011a). Clinical, biological, and skin
manuscript. histopathological effects of ionic macrocyclic and nonionic linear
gadolinium chelates in a rat model of nephrogenic systemic
fibrosis. Invest Radiol 46: 85–93.
Conflicts of interest Fretellier N, Idée JM, Dencausse A, Karroum O, Guerret S, Poveda N

et al. (2011b). comparative in vivo dissociation of gadolinium
chelates in renally impaired rats: a relaxometry study. Invest Radiol
All work was funded by Guerbet. NF, JMI, GJ, CF, NP, AD, NB,
46: 292–300.
MP and CC are or were employees of Guerbet.
George SJ, Webb SM, Abraham JL, Cramer SP (2010). Synchrotron
X-ray analyses demonstrate phosphate-bound gadolinium in skin in
nephrogenic systemic fibrosis. Br J Dermatol 163: 1077–1081.
Gorres KL, Raines RT (2010). Prolyl 4-hydroxylase. Crit Rev
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toxicological sciences 131(1), 259–270 (2013)
doi:10.1093/toxsci/kfs274
Advance Access publication September 12, 2012
Nephrogenic Systemic Fibrosis-Like Effects of Magnetic Resonance

Imaging Contrast Agents in Rats with Adenine-Induced Renal Failure
Nathalie Fretellier,*,†,1 Nejma Bouzian,* Nadège Parmentier,* Patrick Bruneval,‡,§ Gaëlle Jestin,* Cécile Factor,*
Chantal Mandet,‡ Florence Daubiné,¶ France Massicot,† Olivier Laprévote,†,|| Claire Hollenbeck,* Marc Port,*
Jean-Marc Idée,* and Claire Corot*
*Research Division, Guerbet, Roissy Charles de Gaulle Cedex, France; †Chimie Toxicologie Analytique et Cellulaire, Faculté des Sciences Pharmaceutiques et
Biologiques, Université Paris Descartes, Sorbonne Paris Cité, Paris, France; ‡Department of Pathology, Hôpital Européen Georges Pompidou, Paris, France;
§INSERM U970, Department of Pathology, Hôpital Européen Georges Pompidou, Paris, France; ¶Atlantic Bone Screen, Nantes, France; and ||Service de
Toxicologie Biologique, Hôpital Lariboisière, Paris, France
1
To whom correspondence should be addressed at Research Division, Guerbet, BP 57400, Roissy Charles de Gaulle 95943, France. Fax: +33 1 45 91 51 23.
Downloaded from http://toxsci.oxfordjournals.org/ by guest on January 5, 2013

E-mail: nathalie.fretellier@guerbet-group.com.
Received July 18, 2012; accepted September 3, 2012
gadolinium chelates (GCs) used as magnetic resonance imaging

Nephrogenic systemic fibrosis (NSF) is a scleroderma-like contrast agents. This disease has been shown to occur in patients
disease associated with prior administration of certain gado- with severe or end-stage renal disease (ESRD) (Cowper et al.,
linium chelates (GCs). NSF occurs in patients with severe renal 2008). Skin involvement (thickening and hardening of the
failure. The purpose of this study was to set up a rat model of skin associated with plaques, papules, and nodules) is the
GC-associated NSF to elucidate the mechanism of this devastating first sign of the disease. Patients often report pain, associated
disease. Firstly, after characterization of the model, male Wistar with pruritus, paraesthesia, and burning (Cowper et al., 2008).
rats received a 0.75% adenine-enriched diet for 8, 14, or 16 days
Histologically, NSF is characterized by increased dermal
to obtain various degrees of renal failure. Rats received five con-
cellularity, expression of CD34-positive cells with prominent
secutive daily iv injections of saline or gadodiamide (2.5 mmol/kg/
day). Secondly, the safety profile and in vivo propensity to disso- collagen bundles, presence of spindle cells, septal involvement,
ciate of all categories of marketed GCs (gadoterate, gadobutrol, and sometimes osseous metaplasia (Cowper et al., 2008).
gadobenate, gadopentetate, and gadodiamide) were compared in No single diagnostic test is available for NSF. However, a
rats receiving adenine-enriched diet for 16 days. Serial skin biop- working definition including a consensus scoring system has
sies were performed for blinded histopathological study. Total Gd recently been published (Girardi et al., 2011). This definition
concentration in tissues was measured by Inductively Coupled should serve as a working diagnostic standard and the basis for
Plasma Mass Spectrometry. Relaxometry was used to evaluate adjudicating borderline cases. Several authors have reported the
the presence of dissociated Gd in skin and bone. Gadodiamide- involvement of many organ systems referring to the “systemic”
induced high mortality and skin lesions (dermal fibrosis, calcifica- aspect of the disease, which can eventually lead to death (Galan
tion, and inflammation) were related to adenine diet duration. No
et al., 2006).
skin lesions were observed with other molecules. Unlike macrocy-
The recent recognition of a link between GCs and the devel-
clic GCs, gadodiamide, gadopentetate, and gadobenate gradually
increased the r1 relaxivity value, consistent with in vivo dissocia- opment of NSF (Grobner, 2006) has revived the long-debated
tion and release of soluble Gd (or, in the case of gadobenate, the issue of the relevance of the thermodynamic stability of these
consequence of protein binding). Gadodiamide-induced cutane- agents (Morcos, 2008). In order to be used as contrast agents,
ous and systemic toxicity depended on baseline renal function. We the lanthanide element gadolinium must first be chelated with a
demonstrate in vivo dissociation of linear GCs, gadodiamide, and polyaza-polycarboxylic ligand due to its intrinsic toxicity (Idée
gadopentetate, whereas macrocyclic agents remained stable over et al., 2008). Two structurally distinct categories of GCs are
the study period. currently marketed, according to the structure of the ligand:
Key Words: nephrogenic systemic fibrosis; renal impairment; (1) “macrocyclic” chelates (e.g., gadoterate or gadobutrol) in
relaxometry; gadolinium; magnetic resonance imaging; contrast which Gd3+ ions are caged into a cavity of the ligand and (2)
agents.
“linear”, open-chain chelates (e.g., gadopentetate, gadobenate,
or gadodiamide). GC can also be either “nonionic” (where the
number of carboxyl groups is reduced to 3, thereby neutral-
Nephrogenic systemic fibrosis (NSF) is a rare but serious izing the 3 positive charges of Gd3+) or “ionic” (Port et al.,
systemic disease associated with prior administration of certain 2008). GCs dramatically differ in terms of their thermodynamic
© The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For permissions, please email: journals.permissions@oup.com
260 FRETELLIER ET AL.
molecular stability. High kinetic stability (i.e., low dissociation France) for 10 days of acclimatization before starting the experiments. At the
rate over time) provided by the macrocyclic structure combined start of the study (day 0), animals were given a diet containing 0.75% adenine
(SAFE). Rats were housed individually from 1 week after the beginning of the
with high thermodynamic stability minimize the amount of study. All experimental procedures were performed in accordance with French
free Gd3+ that can be gradually released from the parent chelate regulations and in compliance with the European Economic Community
(Port et al., 2008). For lower stability GCs, excess free ligand Directive (2010/63/EU) on animal welfare.
is included in the pharmaceutical preparation to reduce possible
release of Gd3+ during shelf life (Port et al., 2008). The vast Characterization of the Adenine Rat Model
majority of published cases of NSF were associated with the Seven rats received 0.75% adenine-enriched diet for 4 weeks and were then
fed a standard diet (A04) for another 3 weeks. Five rats (normal control group)
nonionic, linear GC gadodiamide and the ionic, less linear GC
were fed a normal diet. The animals were euthanized (exsanguination under
gadopentetate (Rodby, 2011). isoflurane anesthesia) at the end of the experiment (i.e., on day 49 after the start
One of the numerous hypotheses for the mechanism of NSF of adenine diet). Blood (from sublingual vein) and 24-h urine samples were
is that dissociation of less stable GC in at-risk patients may lead collected once or twice a week (days 0, 7, 14, 21, 28, 35, 42, and 49). Blood
to gradual release of the free gadolinium ion Gd3+, which may was used for routine hematological examinations using a MS4-5 automat
(Melet-Schloesing, Osny, France). Plasma levels of total calcium, phosphorus,
subsequently activate trafficking of circulating CD34+ fibro-
transferrin-bound iron, creatinine (Vitros-II auto-analyzer), sodium, chloride,
cytes and/or directly activate resident fibroblasts (Idée et al., and potassium (direct potentiometry, Vitros-350 autoanalyzer, Ortho-Clinical
2008). In vitro proliferative effects of gadolinium have been Diagnostics Inc., Issy les Moulineaux, France) were also measured. Urine lev-
described (Li et al., 2010). An alternative hypothesis is a profi- els of creatinine were determined using a Vitros-II analyzer. Creatinine clear-

brotic effect of the GC molecule itself (Newton and Jiménez, ance (ml/min/100 g bw) was calculated by a standard method.
At necropsy, kidneys and femurs were removed and fixed in 4% neutral
2009). Various rat models of NSF have been proposed to elu-
buffered formalin. After dehydration, the femurs were embedded in glycol
cidate the mechanism of this disease (Sieber et al., 2009). The methacrylate resin, sectioned (4–6 µm thick), and stained with Von Kossa
most common model consists of the administration of GCs in (mineralized bone structure) and Goldner’s Trichrome (osteoid, bone marrow,
subtotally (five sixth) nephrectomized rats (five-sixth SNx) and bone structure) stains. After routine dehydration, the kidneys were embedded
(Fretellier et al., 2011a; Grant et al., 2009; Haylor et al., 2010; in paraffin, sectioned (5 µm thickness), and stained with hematoxylin-eosin-
saffron (HES) for histological examination. Histopathological examinations
Pietsch et al., 2009). Overall, these studies concluded that
were performed in a blinded fashion in accordance with the Society of
fibrotic-like lesions can be induced in rats treated with gado- Toxicologic Pathology guidelines (Crissman et al., 2004).
diamide. Gadodiamide has also been shown to increase Gd
retention in rats, with a higher skin gadolinium concentration Role of Baseline Renal Function on the Clinical and Biochemical
in rats receiving gadodiamide compared with animals receiving Effects of Gadodiamide
gadopentetate. The lowest values were observed in rats treated Study design. Based on the duration of the adenine diet, three levels of
with macrocyclic GCs. However, this interesting and validated renal impairment were tested. Rats received 0.75% adenine-enriched diet for
model presents certain disadvantages such as cost, limited either 8 (study 1), 14 (study 2), or 16 (study 3) days (n = 4–11 rats per group).
Rats were allocated to five consecutive daily iv injections (into the tail vein) of
reduction of glomerular filtration rate (GFR), and absence of 0.5 (study 1) or 2.5 mmol/kg (studies 1, 2, and 3) of gadodiamide (Omniscan,
hyperphosphatemia. Addition of adenine to the diet of rat has GE Healthcare, Batch 11082864 in studies 1 and 3; Batch 10755384 in study
been reported to induce renal failure (Koeda et al., 1988). This 2) or saline from day 7 to 11 (studies 1 and 2) or from day 14 to 18 (study
model has several potential advantages over the five-sixth SNx 3) (Fig. 1). Blood samples were collected for determination of hematological
model (high reproducibility, hyperphosphatemia, and early and biochemical parameters on day 0, the first day of gadodiamide treatment
and at sacrifice. Skin biopsies were performed on the 1st and 8th (study 2) or
drop in GFR with potentially adjustable amplitude). 10th (studies 1 and 3) days of gadodiamide treatment and at sacrifice. Rats
To our knowledge, no study has compared all marketed cat- were euthanized 3 weeks after the last injection of gadodiamide or saline (cor-
egories of GCs in a sensitized (renally impaired) in vivo model. responding to day 32 in studies 1 and 2; day 39 in study 3), and skin, the left
This model would therefore appear to be of great interest. The kidney, and liver were removed.
aim of this study was to (1) characterize this new rat model of Rats were checked for macroscopic skin changes before the first injection
and then daily throughout the study. On the 8th or 10th day of treatment and
NSF, (2) investigate the role of baseline function in rats receiv- at sacrifice, the animals’ backs were carefully shaved to facilitate visualization
ing the nonionic linear GC gadodiamide, and (3) compare the of potential lesions.
safety profile and in vivo propensity to dissociate of all catego-
Histopathological examinations. Skin, kidney, and liver samples were
ries of marketed GCs. fixed in 4% neutral buffered formalin. After dehydration, samples were embed-
ded in paraffin, sectioned (5 µm thickness), and stained with HES stain for his-
tological examination. Immunohistochemistry was performed on skin samples
at sacrifice in studies 2 and 3. Transforming growth factor β1 (TGFβ1), a fibrotic
MATERIALS AND METHODS marker, was detected using anti-TGFβ1 mouse antibody (AbD Serotec, Colmar,
France) and ED-1-positive macrophages (AbD Serotec) were detected using
Animals anti-ED-1 mouse antibody and an antimouse biotinylated antibody (AbCys,
Paris, France) staining. Histopathological examinations were performed, in
A total of 84 male Wistar rats (Centre d’Elevage René Janvier, CERJ, Le blinded fashion, by a certified pathologist.
Genest Saint Isle, France), aged 6 weeks, and weighing 285 ± 39 g at the time
of the study were used. The animals were housed two per cage at an ambient Biochemistry and hematology. Hematological parameters were deter-
temperature of 22°C ± 2°C, hygrometry of 45 ± 10%, with 12/12-h light/dark mined with a MS4-5 automat. Plasma levels of total calcium, phosphorus,
cycles. Rats were given ad libitum access to water and food (A04, SAFE, Augy, blood urea nitrogen (BUN), iron (Vitros DT60II, Ortho-Clinical Diagnostics,
NEW RAT MODEL OF NEPHROGENIC SYSTEMIC FIBROSIS 261
FIG. 1. Experimental design. Adenine–containing diet (0.75%) was administered for 8 days (study 1), 14 days (study 2), or 16 days (study 3) (dark gray bars).
Starting on day 7 (studies 1 and 2) or day 14, rats received five consecutive daily iv injections of saline or gadodiamide (dark cross). The animals were euthanized
25 days after the first injection.
Inc.), creatinine, sodium, chloride, and potassium (Vitros fusion 5.1, Ortho- Bayer Healthcare) for 5 consecutive days starting on day 14 after starting the

Clinical Diagnostics, Inc.) were also determined. These parameters were meas- adenine diet (n = 4 or 6 rats per group). Skin biopsies were performed on days
ured on the first day of gadodiamide treatment (day 7 in studies 1 and 2; day 14, 23, and 39 after the first administration of adenine.
14 in study 3) and at sacrifice. All assays were performed in duplicate. Blood
Histopathological examinations. Histopathological examinations (skin,
samples were collected from the sublingual vein.
kidney, and liver; HES stain) were performed as previously described, in
Relaxometry and gadolinium measurement. Total Gd levels in skin blinded fashion, by a certified pathologist. CD34, ED-1, and TGFβ1-positive
samples were measured by inductively coupled plasma mass spectrometry cells were detected in skin samples.
(ICP-MS) on ELAN DRC Plus (PerkinElmer Life and Analytical Sciences,
Biochemistry and hematology. Blood samples from the sublingual vein
Inc., Waltham, MA) as described elsewhere (Fretellier et al., 2011b). The limit
were collected on days 0, 7, 14, 16, 21, 29, and 39 after starting the adenine
of quantification was 0.64nmol/L.
diet for routine hematological and biochemical examinations. Plasma levels
The presence of dissociated Gd in skin biopsies was assessed by a relax-
of interleukin-1-β (IL1-β), monocyte chemotactic protein (MCP-1), TGFβ1,
ometry technique (in vivo studies), as described elsewhere (Fretellier et al.,
and macrophage inflammatory protein-1 β (MIP-1β) were measured by ELISA
2011b, 2012). Dilutions of 1:11 were performed in D2O/H2O (90/10) mix to
(Bender MedSystems, Vienna, Austria) on days 16 and/or 39. All assays were
mash samples with a Precellys homogenizer. Before measurement, the suspen-
performed in duplicate.
sion was mixed to avoid sedimentation, then checked to ensure a homogene-
ous appearance. Longitudinal relaxation times (T1) were measured on Bruker Relaxometry and gadolinium measurement. In skin, liver, kidney, and
Minispec (Bruker Optics, Ettlingen, Germany) at 60 MHz (i.e., 1.42 T) and at femur samples, total Gd was measured by ICP-MS using an Elan DRC Plus
37°C. When the 1/T1 − 1/T1diamagnetic value was less than 20% of 1/T1diamagnetic in instrument (PerkinElmer Life and Analytical Sciences Inc.). The plasma-dissoci-
the absence of Gd precipitation (i.e., because of a low total Gd concentration in ated Gd3+ concentration was measured by high-pressure liquid chromatography
the sample), it was considered that r1 relaxivity could not be determined. Total linked to an ICP-MS system as described elsewhere (Fretellier et al., 2011b). The
Gd was then determined in samples by ICP-MS. LOQ of the method was 0.5µmol/L.
Relaxivities (in vivo r1 value) were calculated according to the formula: The presence of dissociated Gd in skin biopsies, liver, and femoral epiphysis
r1 = (1/T1sample − 1/T1diamagnetic)/[Gd]sample, with relaxation rate (1/T1) expressed was assessed by relaxometry (in vivo studies). Relaxometry studies (in vitro
in s−1, Gd concentration in mM, and relaxivity r1 in mM−1·s−1. spiking studies) on biological matrices were performed by spiking with GC
Relaxometry studies (in vitro spiking studies) on biological matrices were (from commercial solutions) (range of 0, 0.005, 0.01, 0.02, 0.04, 0.05, 0.1,
performed by spiking with gadodiamide (range of 0, 0.005, 0.01, 0.02, 0.04, 0.5, and 1mM) on D2O/H2O mix, rat skin, femoral epiphysis, and liver from
0.05, 0.1, 0.5, and 1mM) on D2O/H2O mix or rat skin from nontreated rats. nontreated rats as described elsewhere (Fretellier et al., 2011b).
The term “in vitro studies” refers to all experiments performed by spiking
GC on tissue matrices (to obtain the reference range of r1 relaxivities), and Statistical Analyses
“in vivo studies” refers to all experiments performed on test animals. Based
on unpublished in vitro relaxometry studies, the uncertainty of relaxivity r1 Data are expressed as mean ± SD. SD values are given only for n > 2 rats.
measurements (including variability of relaxation time measurement and Gd The effects of test solutions on body weight and biochemical and hematologi-
measurement by ICP-MS) was set at r1 ± 23%. The in vitro r1 relaxivity range cal parameters were compared by ANOVA with repeated measures (effects of
therefore represents the in vitro r1 relaxivity value (obtained from spiking stud- time and treatment). The 95% confidence intervals of in vivo r1 relaxivity were
ies) ± uncertainty of 23%. calculated (mean ± 1.96 SD) and compared with in vitro relaxivity r1 values
± 23% (in vitro r1 range) (Fretellier et al., 2011b). When the confidence inter-
val for mean on the in vivo relaxivity r1 was included in the in vitro relaxiv-
Comparison of All Categories of GCs in Rats Receiving a 16-day
ity r1 range, the in vivo Gd state was considered equivalent to the in vitro
Adenine Diet Gd state (chelated, dissociated, or precipitated), according to the respective
Study design. Rats received 0.75% adenine-enriched diet for 16 days relaxivity r1 value (Fretellier et al., 2011b), or were otherwise considered
(starting on day 0). Rats were allocated to daily intravenous injections of to be different. Assuming a normal distribution of values, Gd concentration
2.5 mmol/kg of gadoterate (Dotarem, batch 10GD54A, Guerbet, Villepinte, measurements were tested globally with ANOVA. In the case of a signifi-
France), gadodiamide (Omniscan, batch 11151244, General Electrics cant difference, pairwise comparisons were performed using a Bonferroni’s
Healthcare, Chalfont St Giles, UK), dimeglumine gadobenate (MultiHance, test. Statistical analyses were carried out using GraphPad Prism software
batch S0P260A, Bracco, Milan, Italy), gadobutrol (Gadovist, batch 04023D, (GraphPad Software Inc., San Diego, CA). Differences were considered sig-
Bayer Healthcare, Berlin, Germany), gadopentetate (Magnevist batch 92083K, nificant for p < 0.05.
RESULTS
Characterization of the Adenine Rat Model

Clinical findings, weight, biochemistry, and hematology. A
decrease in body weight was observed in the group receiving
adenine diet compared with the control group over the adenine
diet period (202.7 ± 22.1 g vs. 432.3 ± 17.1 g, p < 0.05 on day
28). An abrupt decrease in creatinine clearance was observed
in adenine-fed rats from the first week of the adenine period
and creatinine clearance then remained stable until the end of
the study (Fig. 2). A gradually increasing high mortality was
observed in the adenine-fed group from the third week of ade-
nine diet onwards (Fig. 2). An increase in plasma phosphorus
(peak value on day 21: 4.1 ± 1.0 vs. 2.4 ± 0.2 mmol/l in controls, FIG. 2. Time course of creatinine clearance (ml/min/100 g bw) and mor-
p < 0.01) associated with a decrease in plasma total calcium tality in renally impaired (fed with adenine-containing diet for 4 weeks) and
(peak value on day 28: 2.0 ± 0.1 mmol/l vs. 2.5 ± 0.2 mmol/l in control rats. Values are mean ± SD.
controls, p < 0.05) was observed during the adenine period.

An increase in urinary total proteins was observed in adenine- renal failure) (Table 2). In study 1 (low renal impairment),
fed rats (peak value on day 7: 523 ± 144 mg/l vs. 12 ± 4 mg/l in one rat receiving a dose of 2.5 mmol/kg was found dead on
controls, p < 0.01). At necropsy, kidneys of adenine-treated the second day of gadodiamide injection (corresponding to
rats were pale and hypertrophied with a higher mean weight day 8) and in study 2, one rat had to be euthanized on day
than that of kidneys from control rats (1.0 ± 0.05 g/100 g bw vs. 16 for ethical reasons (loss of bodyweight, prostration, and
0.29 ± 0.01 g/100 g bw, p < 0.05). loss of locomotor’s activity). Five of the six rats from study
2 (intermediate renal impairment) presented epidermal skin
Histopathology of kidneys and femurs. Adenine induced lesions (from day 14) and severe ulcerative and squamous skin
major tubular lesions with intratubular lithiasis-like precipitates eruptions were observed in two gadodiamide-treated rats in
and renal interstitial fibrosis with few inflammatory cells (HES) study 3 (i.e., severe renal failure) from day 22. No macroscopic
and no glomerular and vascular changes (Fig. 3A). A marked skin lesions were observed in rats in study 1, corresponding to
decrease in bone tissue mineralization in femoral epiphyses low-grade renal failure.
and/or diaphyseal bone-degrading cell infiltrate was observed
in adenine-fed rats (Von Kossa’s stain) (Fig. 3B). These lesions Histopathology. No histological abnormalities were
were associated with a marked decrease in bone marrow den- observed on skin samples from treated rats in study 1 (Table 2).
sity (Goldner’s Trichrome). Fibrous structures were observed The skin was considered to be abnormal in four of the six
in bone marrow of adenine-fed rats (Fig. 3C). gadodiamide-treated rats in study 2: gradual dermal fibrosis
associated with hypercellularity and/or inflammation. Marked
Role of Baseline Renal Function on the Clinical and foci of early calcification with or without low-grade fibrosis
Biochemical Effects of Gadodiamide and few inflammatory cells were observed in 5 of the 11 gado-
Clinical findings and biochemistry. On day 0 (i.e., before diamide-treated rats in study 3. ED-1 and TGFβ1-positive cells
adenine diet), mean plasma creatinine was 20.9 ± 3.7 µmol/l. were also observed in rats included in study 2 and in the sur-
On the first day of gadodiamide administration, plasma cre- viving rat included in study 3 (Fig. 4). Blinded analysis of the
atinine was equivalent in studies 1 and 2, but was higher in kidneys (and livers) at sacrifice did not reveal any treatment-
study 3 (p < 0.001 vs. studies 1 and 2) (Table 1). At sacrifice, related abnormality in gadodiamide-treated rats. Kidney histo-
plasma creatinine was lower in study 1 than in studies 2 and 3 pathological data were therefore pooled according to the study
(p < 0.05). A decrease in plasma creatinine compared with the (“low,” “medium,” and “high” renal failure) (Table 1).
first day of gadodiamide was observed at sacrifice in studies 1 Major tubular lesions including marked lithiasis, inflamma-
and 3, but remained stable in study 2. tion, and interstitial fibrosis were observed in the kidneys of
On the first day of gadodiamide treatment, the severity of rats from studies 2 and 3, whereas lithiasis was rarely observed
renal failure estimated by BUN was consistent with that esti- in study 1 animals (foci of tubular lesions associated with
mated by plasma creatinine (Table 1). BUN at sacrifice was not inflammation and fibrosis were sometimes observed) (Table 1).
a reliable parameter, as the duration of standard diet differed
between the three studies, and this difference may have inter- Total gadolinium measurement and relaxometry studies.
fered with BUN levels. In all studies, a gradual increase in in vivo r1 relaxivity value,
Gadodiamide administration was associated with high exceeding the in vitro r1 range, was observed in skin samples of
mortality (10/11 rats) in rats in study 3 (i.e., rats with severe gadodiamide-treated rats (Fig. 5).
FIG. 3. Histological findings in kidneys (A) and femurs (B and C) of normal or renally impaired rats. HES stain of kidneys (A), Von Kossa stain (black)/
Ponceau-Fuchsin (pink) (B), and Masson’s Trichrome stain (C) of femurs were performed 49 days after starting adenine diet. In kidneys (A), interstitial fibrosis
associated with a few inflammatory cells was observed in rats fed with adenine diet. Major tubular lesions, with intratubular lithiasis-like precipitate (black arrow)
associated with granuloma, normal glomeruli, and vessels were also observed in this group. In femurs (B), cell infiltrate in the diaphysis (black arrow) degrading
bone surface, and decreased trabeculae were observed in adenine-fed rats. The bone marrow (brown/red) appears less dense compared with physiological bone
and fibrous areas (red arrow) were observed in the trabecular bone and/or diaphyseal bone (C).
TABLE 1
Plasma Creatinine and BUN Levels Measured on the First Day of 2.5 mmol/kg of Gadodiamide Treatment and/or at Sacrifice,
and Histopathology of Kidneys at Sacrifice (HES Stain) in Rats With Low-Grade, Intermediate, or Severe Renal Failure (8, 14, or
16 Days of Adenine-Enriched Diet)
Plasma [creatinine] Plasma [BUN]

(µmol/l) (mmol/l) Histopathology of kidney (number of rats)
Degree of
renal First day of First day of Interstitial
Study failure gadodiamide Sacrifice gadodiamide Grade Lithiasis Tubular lesions Inflammation fibrosis
1 Low-grade Absence 12/15 7/15 7/15 7/15
Day 7: Day 32: Day 7: Mild 1/15 4/15 4/15 4/15
48.2 ± 3.2 21.5 ± 0.7 14.5 ± 3.6 Moderate 1/15 1/15 1/15 1/15
Severe 1/15 3/15 3/15 3/15
2 Intermediate Absence 0/10 0/10 0/10 0/10
Day 7: Day 32: Day 7: Mild 0/10 0/10 0/10 0/10
59.3 ± 7.2 66.6 ± 10.2 14.9 ± 6.6 Moderate 1/10 1/10 1/10 1/10
Severe 9/10 9/10 9/10 9/10
3 Severe Absence 2/5 0/5 0/5 0/5

Day 14: Day 39: Day 14: Mild 0/5 0/5 0/5 0/5
139.9 ± 11.7 56.9 ± 12.1 25.6 ± 4.2 Moderate 0/5 0/5 0/5 0/5
Severe 3/5 5/5 5/5 5/5
Statistical analysis Study 3 vs. Study 1 vs. Study 3 vs. Study 1 vs. study Study 1 vs. study Study 1 vs. study Study 1 vs. study
studies 1 and studies 2 and studies 1 and 2, p < 0.001 2, p < 0.01 2, p < 0.01 2, p < 0.01
2, p < 0.001 3, p < 0.05 2, p < 0.001 Study 1 vs. study Study 1 vs. study Study 1 vs. study Study 1 vs. study
3, p < 0.05 3, p < 0.05 3, p < 0.05 3, p < 0.05
Note. “Mild lesions” corresponding to lesions < 25% of slide surface; “moderate” lesions corresponding to lesions between 25 and 50% of slide surface;
“severe” lesions corresponding to lesions > 50% of slide surface.
TABLE 2
Clinical Signs and Skin Histopathology After Treatment With Gadodiamide in Rats With Low-Grade, Intermediate, or Severe Renal
Failure (8, 14, or 16 Days of Adenine-Enriched Diet)
Morbidity/Macroscopic
Study Gadodiamide dose N (rats) Mortality skin lesions Skin histopathology
1 (low-grade renal failure) 5 × 1.0 mmol/kg 6 No No No abnormalities
5 × 2.5 mmol/kg 6 1 found dead(day 8) No No abnormalities
Saline 4 0/4 No No abnormalities
2 (intermediate renal failure) 5 × 2.5 mmol/kg 6 1 euthanized 4/5: skin lesions (2: ++) Two rats: no lesions
(Day 16) Euthanized rat: minor Four rats (including euthanized rat):
abdominal lesions hypercellularity TGFβ+ and inflamma-
tory foci (ED-1-positive macrophages)
Saline 4 0 No No abnormalities
3 (severe renal failure) 5 × 2.5 mmol/kg 11 10 Prostration, piloerection, Five rats: Low-grade fibrosis at the time
loss of body weight, of euthanasia, few inflammatory foci,
and skin lesions (two and TGFβ+ immunostaining. Early
rats) calcification foci
Saline 5 0 No No abnormalities
Total gadolinium concentration in skin at sacrifice (25 days linear correlation was observed between skin total Gd and
after the first injection of gadodiamide) was higher in the single plasma creatinine concentrations (Fig. 6).
surviving rat from study 3 (216.6 nmol/g) than in rats included
in study 2 (168.4 ± 105.2 nmol/g) and rats with low-grade renal Comparison of All Categories of GCs in Rats Receiving
failure (69.0 ± 43.1 nmol/g). Interestingly, in Study 2, the skin a 16-Day Adenine Diet
total Gd concentration measured in the only rat without skin Clinical findings. Three of four gadodiamide-treated rats
lesions was around 7-fold lower than in the rats with skin were either found dead or had to be euthanized for ethical rea-
lesions (31.3 vs. 202.7 ± 83.1 nmol/g). A significant positive sons (loss of bodyweight > 20%, prostration, and marked loss of

FIG. 4. Histological skin lesions in the dermis: HES staining to analyze cellular lesions, TGFβ1 staining, and ED-1 staining to detect macrophages were
performed at the end of the studies (day 32 for studies 1 and 2; day 39 for study 3).
FIG. 5. In vivo relaxivity r1 values (60 MHz, 37°C) in skin samples from
rats fed an adenine diet for 8 (study 1), 14 (study 2), or 16 (study 3) days and
treated with five consecutive daily injections of gadodiamide (2.5 mmol/kg).
Gray bars correspond to in vitro r1 range of gadodiamide in dorsal skin.
FIG. 6. Positive linear correlation between plasma creatinine and skin gad-
locomotor activity). Transient epidermal lesions were observed olinium concentration at sacrifice in rats receiving five consecutive daily injec-
between day 17 and 18 on the abdomen of four of the six tions of gadodiamide (2.5 mmol/kg) following adenine diet for either 8 days
(study 1) or 14 days (study 2). Dotted lines indicate 95% confidence limits.
gadopentetate-treated rats. No macroscopic skin lesions were
observed for other treatments except for wrinkled skin in the
gadodiamide-treated survivor. No significant difference in body sodium, potassium, chloride, phosphorus, calcium, and iron
weight changes was observed between the test groups. One rat levels or hematological parameters (data not shown).
in the gadoterate and gadobenate-treated groups died, but death On day 16, plasma MCP-1 levels were significantly higher
was unrelated to injection of the product (due to anesthesia). (13.2 ± 1.8 pg/ml vs. 4.4 ± 1.4 pg/ml in the control group;
p < 0.001 vs. other groups) for gadodiamide-treated rats.
Biochemistry and hematology. No significant differences However, no differences between treatments were observed on
were observed between groups in terms of plasma creatinine, day 39. No significant changes in the plasma levels of the other
FIG. 7. Total Gd concentration measured in skin samples of rats fed an adenine diet for 16 days and receiving each GC (5 × 2.5 mmol/kg iv from day 14 to
18) on days 23 and 39 (i.e., sacrifice) after starting adenine diet (ICP-MS measurement, nmol/g). Mean and individual values are given. ***p < 0.001 vs. all groups.

FIG. 8. Total Gd concentration measured in femur samples (epiphyses) of rats fed an adenine diet for 16 days and receiving each GC (5 × 2.5 mmol/kg iv from
day 14 to 18) on day 39 (i.e., sacrifice) after starting adenine diet (ICP-MS measurement, nmol/g). Mean and individual values are given. ***p < 0.001 vs. all groups.
cytokines and the enzyme TIMP-1 were observed, regardless of Skin total gadolinium levels. No significant differences in
the GC tested (data not shown). skin total Gd concentration on day 23 were observed between
treatment groups, except for gadobenate-treated rats, in which
Histopathology. Skin biopsies were studied on days 14, 23, the total Gd concentration was significantly lower than in the
and 39. No abnormalities were observed on skin biopsies from other groups (p < 0.001). A dramatic decrease in the skin total
treated groups except for the gadodiamide-treated survivor rat, Gd concentration on day 39 (vs. day 23) was observed in the
in which mild inflammation of the dermis without fibrosis was gadoterate, gadopentetate, gadobenate, and gadobutrol-treated
observed at day 39. Positive immunostaining for TGFβ1, TIMP-1, groups. The total Gd concentration in dorsal skin on day 39 was
and ED-1 was observed in the dermis of the gadodiamide-treated higher in the gadodiamide-treated survivor (128.8 nmol/g) than
survivor at sacrifice. No increase in the density of CD34- and pro- in the other GC-treated groups (Fig. 7).
lyl-4-hydroxylase-positive cells was observed in the dermis, what-
ever the groups. At sacrifice, no abnormalities were observed in Total gadolinium levels in the femur. Total Gd concentra-
the lungs, kidney, or liver samples in any of the treatment groups. tions in the femur on day 39 were higher in the gadopentetate-
treated groups than in the gadoterate, gadobenate, and
Gadolinium Measurement gadobutrol groups (p < 0.001) (Fig. 8). A high total Gd con-
Plasma-dissociated Gd3+ levels. The plasma Gd3+ concen- centration was measured in the gadodiamide-treated survivor
tration at day 21 was below the limit of detection for the ionic (207.8 nmol/g).
macrocyclic GC gadoterate and the nonionic macrocyclic GC
gadobutrol. At this time point, it was 79.3µM in the single gado- Total gadolinium levels in kidneys and liver. No signifi-
diamide-treated survivor. In one rat from the gadobenate and cant differences in total Gd concentrations in the kidneys
gadopentetate-treated groups with measurable Gd3+ levels, the dis- and liver were observed between treatment groups (data not
sociated Gd3+ concentrations were 0.44 and 3.0µM, respectively. shown).
TABLE 3
In Vitro r1 Relaxivity Value (Spiking Studies With Gadoterate, Gadodiamide, Gadopentetate, Gadobutrol, or Gadobenate [60 MHz,
37°C]) in D2O/H2O and Various Tissue Matrices From Control Rats
In vitro r1 relaxivity (mM−1·s−1) [± 23%]
Matrix Gadoterate Gadopentetate Gadobenate Gadobutrol Gadodiamide

D2O/H2O (90/10 vol/vol) 3.3 3.8 4.8 3.8 3.7
[2.5–4.1] [2.9–4.7] [3.7–5.9] [2.9–4.7] [2.9–4.6]
Femoral epiphysis 3.8 4.3 4.6 4.5 4.6
[2.9–4.7] [3.3–5.3] [3.5–5.7] [3.5–5.6] [3.5–5.7]
Dorsal skin 3.0 3.6 4.8 3.5 4.5
[2.3–3.7] [2.8–4.4] [3.7–5.9] [2.7–4.3] [3.5–5.6]
Liver 4.1 4.4 5.7 4.3 4.0
[3.2–5.0] [3.4–5.5] [4.4–7.0] [3.3–5.4] [3.1–4.9]
Note. The uncertainty of relaxivity r1 measurements was set at r1 ± 23%.

FIG. 9. In vivo relaxivity r1 values (60 MHz, 37°C) in skin samples of rats fed an adenine diet for 16 days and treated with each GC (5 × 2.5 mmol/kg) on day
23 or 39 (i.e., sacrifice) after starting adenine diet. Mean and individual values are given. Gray bars correspond to in vitro r1 range of each GC in dorsal skin (r1 ±
23%). **p < 0.01 between day 14 and day 39 in gadopentetate-treated group. LOQ, limit of quantification.
Relaxometry Studies and gadodiamide groups due to the insufficient number of

The in vitro r1 relaxivity values obtained in D2O/H2O, skin, animals.
liver, and femoral epiphysis matrices are shown in Table 3.
Relaxometry values in the femur. The in vivo r1 relaxivity
Relaxometry values in the skin. In in vivo studies, the values in the femoral epiphysis were higher in the gadobenate
skin r1 relaxivity value increased from 4.0 ± 0.1mM−1·s−1 on group (15.9mM−1·s−1, two rats with measurable levels) and
day 14 to 10.6 ± 3.8mM−1·s−1 on day 39 in the gadopentetate gadopentetate group (8.8 ± 1.8mM−1·s−1), than in the gadoter-
group (p < 0.01), and from 5.1 ± 0.2mM−1·s−1 on day 14 to ate group (no rats with calculable r1 value, as the 1/T1 − 1/
11.1mM−1·s−1 (two rats had measurable r1 values) on day 23 in T1diamagnetic value less than 20% of 1/T1diamagnetic due to a low total
the gadodiamide group (p < 0.1). The skin r1 values on days 23 Gd concentration). One rat in the gadobutrol-treated group had
and 39 were higher in the gadodiamide group than the skin r1 a measurable r1 value (6.2mM−1·s−1). The r1 relaxivity values
values on day 14, whereas no significant changes were observed measured in femoral epiphysis samples of the gadopentetate
in the other groups (Fig. 9). group were higher than those of the in vitro range (Fig. 10A).
The r1 relaxivity values measured in skin samples of the
gadopentetate group were higher than those of the in vitro Relaxometry values in the liver. No significant differences
range on days 23 and 39. The r1 relaxivity values measured were observed between groups in terms of in vivo r1 value in the
in skin samples of the gadoterate and gadobutrol groups were liver. The in vivo r1 relaxivity values measured in liver samples
situated within the in vitro range, but no conclusions could from the gadopentetate and gadobenate groups were higher
be reached for the r1 values measured in the gadobenate than the respective in vitro ranges (Fig. 10B).

FIG. 10. In vivo relaxivity r1 values (60 MHz, 37°C) in femoral epiphysis (A) or liver (B) samples of rats fed an adenine diet for 16 days and treated with
each GC (5 × 2.5 mmol/kg) on day 39 (i.e., sacrifice) after starting adenine diet. Values are mean and individual values. Gray bars correspond to in vitro r1 range
of each GC in femur (A) or liver (B) (r1 ± 23%). LOQ indicates limit of quantification.
DISCUSSION fibrosa) was observed under our study conditions. Osteitis fibrosa
is common in patients with chronic renal failure (Coladonato,
Clinically relevant preclinical models are crucial to eluci- 2005). The most severe renal impairment was observed when
date the mechanism of this devastating disease. The five-sixth the adenine-containing diet was given for 4 weeks. However,
SNx rat model is frequently used to mimic human renal failure progressively increasing mortality was also observed, making a
(Fretellier et al., 2011a; Grant et al., 2009; Haylor et al., 2010; 4-week protocol unsuitable for further studies.
Pietsch et al., 2009). This study used the adenine-enriched We subsequently investigated the relationship between the
model of renal failure in rats, developed by Yokozawa et al. amplitude of renal failure and the clinical effects of gadodi-
(1986). Basically, adenine is converted to AMP. However, in the amide. Most reported cases of NSF are associated with this
presence of excess of adenine, an alternative pathway is acti- agent (Rodby, 2011). High mortality and macroscopic skin
vated, leading to 2.8-dihydroxyadenine urolithiasis and eventu- lesions were observed in two rats with severe renal impair-
ally to renal failure (Koeda et al., 1988; Okada et al., 1999). ment. Microscopic lesions (i.e., fibrosis and calcification) were
Modulation of the duration of adenine diet resulted in various observed in 5 of the 11 rats from this group. In rats with inter-
degrees of renal failure, allowing maintenance of renal func- mediate renal failure, the majority of rats developed ulcerative
tion for a defined period, in agreement with Okada et al. (1999). and squamous skin eruptions associated with fibrosis, hyper-
Hyperphosphatemia associated with osteodystrophy (osteitis cellularity, and inflammation. Overexpression of the profibrotic
marker TGFβ1, the collagenase inhibitor TIMP-1, and an of gadopentetate-treated rats. In rats, 50% of gadobenate is
increased number of ED-1-positive macrophages in the skin excreted by the liver (Lorusso et al., 1999), unlike other GCs
were observed. These findings are in agreement with a previ- which are excreted exclusively by the kidneys (Idée et al.,
ously published report (Fretellier et al., 2012) and with those 2009). This may explain the relatively low total Gd concentra-
observed in NSF patients (Jiménez et al., 2004; Kelly et al., tions found in tissues in gadobenate-treated rats.
2008, 2010). No toxicity was observed in rats with low-grade To our knowledge, this is the first study to compare the in vivo
renal impairment. Under these conditions, skin profibrotic stability of all categories of GCs. Relaxometry can be used to
lesions and systemic toxicity (including mortality) induced by determine longitudinal relaxation rates of tissues. Gadolinium,
gadodiamide were therefore inversely correlated with baseline present in paramagnetic GCs, accelerates relaxation times
renal function. These findings are clinically relevant because (Caravan et al., 1999). Relaxometry is therefore a useful tool to
the majority of cases have occurred in patients with ESRD, and investigate the in vivo dissociated vs. chelated state of Gd fol-
about 20% were reported in patients with acute kidney injury or lowing systemic administration of CG (Fretellier et al., 2011b).
stage 4 chronic kidney disease (CKD) (Abu-Alfa, 2011). There Our results suggest gradual in vivo dissociation, with release of
have been no definitive NSF cases reported in patients with soluble Gd3+ from gadodiamide and from the linear, ionic GC
stage 3 CKD to date, with the exception of one possible case gadopentetate, whereas the more stable macrocyclic GCs gad-
(Abu-Alfa, 2011). Pietsch et al. (2009) reported that renal fail- oterate and gadobutrol remained stable throughout the study. In
ure in rats induced prolonged GC circulation time, as observed vivo dissociation of gadodiamide has already been reported in

in patients with severe renal impairment, which is consistent the literature (Fretellier et al., 2011b, 2012), but in vivo disso-
with the positive correlation between plasma creatinine levels ciation of gadopentetate has never been previously reported, to
and skin total Gd concentrations found in this study and else- our knowledge. The ionic and linear GC, gadobenate, induced
where (Haylor et al., 2010). Pietsch et al. also reported skin increased r1 relaxivity values in the skin (day 23) and femur,
lesions in rats receiving gadodiamide. Interestingly, lesions which may suggest gradual in vivo dissociation of this com-
occurred in the animals with the highest skin Gd concentration. pound, but which would also be consistent with protein binding
We observed similar findings: the only rat without skin lesions (4%) which is known to increase the r1 relaxivity value (Port
and intermediate renal failure had a lower skin Gd concentra- et al., 2005).
tion than rats with macroscopic skin lesions. Our findings sup- We therefore report in vivo dissociation of linear GCs, gado-
port the hypothesis that skin lesions may be dependent on the pentetate and gadodiamide, with gradual release of soluble, disso-
amount of Gd retained in the tissues. Interestingly, High et al. ciated, Gd3+. Macrocyclic GCs have higher kinetic stability than
(2010) showed gradients of Gd deposition correlated to fibrosis linear GCs (Port et al., 2008), as demonstrated directly (Frenzel
and hypercellularity in the skin of two NSF patients. et al., 2008) or indirectly (Laurent et al., 2001). Free Gd3+ has
The purpose of our third study was to compare all categories been reported to induce fibroblast proliferation (Li et al., 2010),
of GCs in rats with impaired renal function. High mortality was but our data do not allow us to draw conclusions on a causal link
observed after administration of gadodiamide, consistent with between dissociated Gd3+ and skin lesions and mortality.
the results of our first study. However, no skin lesions were In summary, cutaneous and systemic toxicity induced by
observed in this group, in contrast with the literature (Grant gadodiamide depends on baseline renal function in an adenine
et al., 2009; Fretellier et al., 2011a, 2012; Pietsch et al., 2009). rat model of NSF. In vivo dissociation of gadodiamide with
This discrepancy can be explained by early death of the ani- gradual release of soluble Gd3+ was observed, whereas macro-
mals. Transient punctate skin lesions were found in four out cyclic agents remained stable over the study period. For the first
of six gadopentetate-treated rats during the injection week. To time, gradual dissociation was suspected for the ionic, linear
our knowledge, this has never been previously reported. It is GC gadopentetate. Further studies are required to investigate
noteworthy that gadopentetate, a linear, ionic GC, like gado- the molecular potential effect of nonchelated Gd3+.
diamide, belongs to the high-risk category of GCs according
to both the European Medicines Agency and the FDA (Rodby,
2011; Thomsen, 2011). However, no histopathological lesions ACKNOWLEDGMENTS
were observed in this group. The first skin biopsy was per-
The authors thank Drs Anthony Saul and Dominique Debize-
formed 5 days after the last GC injection, which might explain
Henderson for reviewing the English version of the manuscript
the absence of histological lesions; epidermal lesions were
and to Charlotte Gary for helpful discussions. N.F., J.M.I., G.J.,
transient and occurred earlier.
C.F., N.P., N.B., C.H., M.P. and C.C. are or were employees of
Skin Gd levels gradually declined in all groups except in the
Guerbet.
gadodiamide group, in which the survivor had a high skin total
Gd concentration. High Gd levels have already been reported
for this compound in the skin (Pietsch et al., 2009; Sieber et al., FUNDING
2008) and in the femur (Sieber et al., 2008). Interestingly, a
high total Gd concentration was also observed in the femur The work was supported by Guerbet.
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7.5
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5.0
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2.5
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F 92 - 23B
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Avec lésions
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Phosphatémie
(mmol/L)
3
0
J0 J7 J21 J32
<< % A - 23
5 E 98 F 9
9. F 92 B
7
6 %\ ] A- 3A
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Phosphatémie
5
(mmol/L)
4
3
2
1
0
0 5 10 15 20 25
Score clinique
<= % F 92 - 2" G 43B
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92 B
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450
400 . W A-AB
350
(nmol/g) à 32
300
250
200
150
100
50
0
-50 5 10 15 20 25 30
-100 Score clinique
= % !
F 9'2B
=2 % F 92 922
5 E 98 F 9
9. F 92
500 %\ ] A- C
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(nmol/g) à J21/22
400
300
200
100
0
0 5 10 15 20 25
Score clinique
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Avant traitement
Gadodiamide + Paclitaxel
Gadodiamide
Période traitement Paclitaxel
25
20
Score clinique individuel
15
10
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Jours
=> % A -98 F 9 3
-9'> F 95=3 - 53
>>H
H Q
Avant traitement
Gadodiamide + DMSO
Gadodiamide
25 Période traitement DMSO
20
15
10
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Jours
=8 % A -98 F 9 3
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, )9($ 42 =<41
3500
NaCl 0,9% + DMSO
cellules/mm² dans le
3000
NaCl 0,9 % + Paclitaxel
2500 gadodiamide + DMSO
Nombre de
derme
2000 gadodiamide + Paclitaxel

1500
1000
500
0
J7 J36 J58
=< % " F 98 - ( ."=@ 3"
9'> - *J , 3 94< - 3 B
3000
NaCl 0,9%
cellules/mm² dans le
XX Gadodiamide
Nombre de
2000
derme
1000
0
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NaCl 0,9 % + DMSO NaCl 0,9 % + paclitaxel

25 25
*
Neutrophiles (%)
Neutrophiles (%)
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15 15
10 10
5 5
0 0
Avant traitement Après traitement Avant traitement Après traitement
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[Gd] total dans la peau
600
550 NaCl 0,9 % + Paclitaxel
500
(nmol/g)

350
300
250
200
150
100
50
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2500 NaCl 0,9% + DMSO

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(nmol/g) à J58
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1500
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500
400
300
200
100
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Avant traitement
Gadodiamide + Paclitaxel
Gadodiamide
Période traitement Paclitaxel
25
20
15
10
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Jours
.8 % A E
- 53
Avant traitement
Gadodiamide + DMSO
Gadodiamide
25
Période traitement DMSO
20
15
10
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Jours
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Créatinémie à J18
175
(µmol/L)
150
125
100
0 5 10 15 20 25
Score clinique
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900
800 NaCl 0,9% + DMSO
(nmol/g)

400
300
200
100
0
J18 J25 J32
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[Gd] total dans la peau dorsale à J18
1000 %\ ] A- CH
. W A-A
750
(nmol/g)
500
250
0
110 120 130 140 150 160 170 180 190 200
Créatinémie à J18 (µmol/L)
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900 . W A-A
800
700
(nmol/g)
600
500
400
300
0 5 10 15 20 25
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9 <
Inflammation Fibrose β
TGFβ
25 25 25
20 20 20
Score clinique
Score clinique
Score clinique
15 15 15
10 10 10
5 5 5
0 0 0
- + ++ +++ - + ++ +++ - + ++ +++
Score histologique Score histologique Score histologique
9'2
Inflammation Fibrose β
TGFβ
25 25 25
20 20 20
Score clinique
Score clinique
Score clinique
15 15 15
10 10 10
5 5 5
0 0 0
- + ++ +++ - + ++ +++ - + ++ +++
Score histologique Score histologique Score histologique
8% - " 3
&! O P E
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Gadolinium Et Fibrose

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Gadolinium Et Fibrose

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Rôle des complexes de gadolinium dans le mécanisme de

la fibrose systémique néphrogénique

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HAL Id: tel-00843108

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