Physical Basis of Gadolinium Induced Skin Nephrofibrosis

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Article ID: WMC003089 ISSN 2046-1690

Physical Basis of Gadolinium Induced Skin

Nephrofibrosis: Testing by Gadolinium-Protein

Targeting Assay and Iron Oxide Nanoparticle Based

Magnetic Resonance MicroscopyCorresponding Author:

Prof. Rakesh Sharma,Professor, Amity Institute of Nanotechnology, Amity University, Sector 125, Express Highway, NOIDA, UP,

201303 - India

Submitting Author:

Prof. Rakesh Sharma,

Professor, Amity Institute of Nanotechnology, Amity University, Sector 125, Express Highway, NOIDA, UP,

201303 - India

Article ID:WMC003089

Article Type:Original Articles

Submitted on:06-Mar-2013, 11:34:48 AM GMT Published on:06-Mar-2013, 12:35:57 PM GMTArticle URL:http://www.webmedcentral.com/article_view/3089

Subject Categories:TOXICOLOGY

Keywords:Skin, Kidney, MRI, 500 MHz NMR, 21 Tesla MR microscopy, Spectroscopy, Gadolinium,

Nephrogenic Systemic Fibrosis

How to cite the article:Sharma R. Physical Basis of Gadolinium Induced Skin Nephrofibrosis: Testing by

Gadolinium-Protein Targeting Assay and Iron Oxide Nanoparticle Based Magnetic Resonance Microscopy .

WebmedCentral TOXICOLOGY 2013;4(3):WMC003089

Copyright:This is an open-access article distributed under the terms of the Creative Commons Attribution

License(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the

original author and source are credited.

Source(s) of Funding:

None

Competing Interests:

Author does not have any conflict and any competing interests

Additional Files:

RAKESH SHARMA- Nano contrast imaging agent, Gadoli

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Physical Basis of Gadolinium Induced Skin

Nephrofibrosis: Testing by Gadolinium-Protein

Targeting Assay and Iron Oxide Nanoparticle Based

Magnetic Resonance MicroscopyAuthor(s):Sharma R

Abstract

Gadolinium based MRI contrast agents are functional

and cationic morphometric markers but toxic to cause

undefined fibrosis in skin and kidney damage.

Magnetic Resonance Microimaging of rat skin andkidney was used first time to identify the physical

factors modulating the gadolinium Omniscan induced

fibrosis by protein targeting.

Hypothesis:Gadolinium contrast agent containing

less chelated endogenous ions target Gd-protein

interactions in both epidermal thickening of skin with

result of dermatopathy and renal basement membrane

proteins with result of nephrofibrosis.

Materials and Methods:Gadolinium contrast agent

was injected in rat animal. 500 MHz MR imaging was

done to visualize fibrosis in gadolinium treated animals.

In other alternative method to enhance the MR imagecontrast, cationic superparamagnetic iron oxide

magnetoferritin (SPIOM) was injected in rat to target

basement membrane(in rat kidney and different skin

structures including epidermis glycolipids and dermis

proteins. After MRI imaging, excised rat skin and

kidneys tissues were imaged by ex vivo 900 MHz MR

microimaging to confirm renal fibrosis and skin

epidermis thickening.

Results: Phantom showed change in magnetic

resonance signal intensity dependence upon protein

and GdIII concentration. Stereotactic arrangement of

coordinate bonds between GdIII-ligand and protein

was associated with relaxivities. The proton density

weighted images visualized micro details of skin

structures and nephron territories while T2 weighted

images showed better contrast of tissue structures in

both skin and kidney. The Gadolinium further

enhanced the image contrast and targeted the

proteins in renal basement membrane and viable

proteins in epidermis. SPIOM enhanced the tissue

contrast due to dephasing effect caused by SPIOM on

structural changes in nephron and epidermis.

Conclusion: Tissue membrane protein and chelate

ligand group binding with gadolinium biophysical

interaction at molecular level may develop fibrosis and

dermatopathy. SPIOM injection improved the

dephased image contrast of different structures in both

skin and nephrons. The epidermis thickening and

nephrofibrosis changes may be associated with

nephrogenic systemic f ibrosis or f ibrosing

dermatopathy.

Key words:Skin, Kidney, MRI, 500 MHz NMR, 21

Tesla MR microscopy, Spectroscopy, Gadolinium,

Nephrogenic systemic fibrosis.

Introduction

Gadolinium (Gd) based compounds are routinely used

as imaging contrast enhancing agents in dynamic

magnetic resonance imaging and angiography (MRI

and MRA) to evaluate function of kidney.

Gadolinium-enhanced MRI or MRA studies frequently

showed Nephogenic Systemic Fibrosis (NSF)

associated with skin thickening, endothelial damage or

thrombosis due to hypercoagulation is public health

concern [1] which still remains an open question to

solve in future [2]. Gd bound to complex ligand

chelating open chain or macrocyclic molecules (GBCA)

enhance the imaging qualities while facilitating its safe

transit and exit from the body. GBCA are excreted out

via the kidneys. In normal kidney function, use of

macrocyclic GBCA imaging agents are safe because

of strong bond formation between the toxic Gd atom

and its kinetically inert (kobs) cyclic ligand molecule.

GBCA molecule is flushed from the kidney rapidly. Incase of delayed excretion, Gd may bind to phosphate

bound molecules flowing in the circulation to form

insoluble Gd molecules. These insoluble Gd

molecules are not readily removed from the body and

accumulate. In patients with kidney disease,

administered GBCA requires more time to exit out

from the body due to delayed excretion. Common

Gadolinium MRI contrast agents are Omniscan 0.5

m o l / l i t e r ( g a d o d i a m i d e ;

gadoliniumdiethylenetriamine-pentaacetate-bismethyla

mide[Gd-DTPA-BMA], Magnevist 0.5 mol/liter

(gadopentetate dimeglumine; Gd-DTPA), ProHance0.5 mol/liter (gadoteridol; Gd-HP-DO3A), MultiHance



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0.5 mol/liter (gadobenate dimeglumine; Gd-BOPTA),

and OptiMARK 0.5 mol/liter (gadoversetamide;

Gd-DTPA-BMEA) are approved by the U.S. Food and

Drug Administration (FDA), others Dotarem 0.5

mol/liter (gadoterate meglumine; Gd-DOTA),

Primovist 0.25 mol/liter (gadoxetic acid disodium;

Gd-EOB-DTPA), Gadovist 0.5 mol/liter (gadobutrol;

Gd-BTDO3A) and Vasovist 0.25 mol/ l i ter

(gadofosveset trisodium; MS-325; diphenylcyclohexyl

phosphonooxymethyl -Gd-DTPA). The gadolinium

chelate contains caldiamide. If Gd chelate dissociates

or dechelates, it may facilitate the increasing number

of circulating fibrocytes to trigger NSF. The stability of

Gd imaging contrast agents is in following order

(Magnevist > Prohance or Multihance > OptiMARK >

Omniscan). Recent studies have reported Gd

deposition and several biophysical factors (pH, pO2,enzymes) of gadolinium bound chelates in imaging

agents (interaction of chelate with tissue proteins) may

be responsible for increased proton relaxation in skin

dermis, alteration of the normal collagen I/III bundles

and increased dermal deposits of mucin in dermis with

NSF associated skin and renal tissue changes

associated with gadolinium toxicity [8,9] as reviewed in

Table 1 [2-16]. Other biophysical factors may be

gadolinium stability and dissociation, conformational

physical factors, ionic factors [17-22].

Gadolinium and Origin of Nephrogenic FibrosingDermopathy:

Nephrogenic Systemic Fibrosis (NSF) was reported a

new entity associated with nephrogenic fibrosing

dermopathy (NFD) in people with kidney disease due

to Gd induced fibrosis[1,2,3]. NSF was reported with

visible physical symptoms of the skin disorder or

dermopathy. Previous kidney disease may also

develop nephrogenic disorder or NSF like symptoms.

Recently, NSF symptoms were reported after MRI

imaging as associated side effects after the use of

gadolinium-based MRI contrast agents in patients with

kidney disease [2,7,8]. Exact mechanism of fibrosisdue to gadolinium is not known. Extracellular Gd

imaging contrast agent is eliminated from the body by

normal kidneys. Patients with renal impairment show

delayed gadolinium excretion due to longer stay and

prolonged biological half-life of less stable gadolinium

molecules and increase the possibility of Gd

transmetallation. Both these factors combined with

endogenous protein ions bound with Gd contrast

agent may further delay the release of free Gd from

the body [6, 15]. Free Gd gets deposited in peripheral

tissues and free Gd may target circulating fibrocytes to

initiate the process of fibrosis. In addition, Gd chelates

in renal tissues may also cause the increased release

of a variety of proteins such as cytokines, transforming

growth factor beta (TGF ), activation of the enzyme

transglutaminase 2 (TG2), receptor induced

magnetization enhancement of galactosidase, alkaline

phosphatase, carboxypeptidase, carbonic anhydrase,

myeloperoxidase proteins to promote fibrosis [22,23].

Free gadolinium molecules get deposited at two major

sites: skin in affected areas and renal basement

membrane in patients to develop NSF [24].

MRI visible microstructures in Kidneys and Skin:

Previous study reported stratum corneum rich in

glycolipids and hairs rich in keratin as distinct and

measurable structures (up to 15 microns) at 900 MHz

MRI as shown in Figure 2A[25]. Two vital regions in

kidneys are MRI visible: cortex and medulla tissues at

low resolution. At high resolution 900 MHz MRI, eachnephron microstructure is indicated as made of single

glomerulus and nephron tubule. Histology showed

further each glomerulus containing fine capillaries. The

glomerular capillary tuft enhances filtration over large

surface area in nephron. The MRI visible filtration unit

in nephron (in suprarenal cortex) is made of

fenestrated epithelial cells, GBM, and podocytes (see

Figure 2B).[26] The semi permeable filters or GBM

barrier layer in nephron retain anionic large proteins in

the blood. In nephron, epithelial cells line up with

capillaries which show distinct fenestrations (30-100

nm) as openings into proteoglycan rich anionic GBMbarrier. Podocyte foot processes on capillaries form

intracellular filtration slits to form a secondary site to

filter proteins [16]. The proton rich solutes and proteins

are MRI visible by using gadolinium contrast agent. In

in vivo MRI, proteins serve as renal filtration rate

biomarker to measure GFR using gadolinium contrast

enhancement [27]. Dynamic magnetic resonance

imaging (D-MRI) was recently reported to evaluate the

renal basement membrane and renal function in

compromised proteinuria state [16,27].

Physical Principles of MR Microimaging:21.1 Tesla MR Microimaging a technological

development: Currently, 21.1 Tesla MR microimaging

magnet operable at 900 mHz is available for achieving

spatial resolution up to 15 micron [26]. The imager has

specific design: 1. magnet employs Nb3Sn and NbTi

conductors in a set of epoxy impregnated long

solenoids plus compensation coils for magnetic field

uniformity to store large magnetic energy with

permeability limit of 900 MHz magnet /0 =1.020 at

operating temperature 1.8 K; 2. cold bore size 138

mm with maximum outer diameter, 878 mm of

windings, maximum height of windings 1500 mm,

weight of Nb3Sn conductor coil 921 kg, weight of



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Nb3Sn reinforcement coil 593 kg, weight of NbTi

conductor coil 1273 kg, weight of NbTi reinforcement

coil 824 Kg at operating current of 290 A; inductance

of coils 953 H; stored energy 40 MJ; permeability limit

for the 900 MHz magnet /0 =1.020; 3. bucking coils

have Nb3Sn-NbTi joints; 4. coils wound as single core

conductor connected through NbTi leads with

intermediate NbTi-NbTi joint; 5. superconducting high

temperature superconductor (HTS) at operating

temperature 80 K-10 K; 6. power supply equipped with

a quench detector, a circuit breaker switch; volume

fraction of the coil 0.15 - 0.55 cm; 7. automated

quench protection by an external dump resistor and

quench detector sensor and a protective circuit of

inductor and resistor; 8. liquid Helium storage at the

top of magnet coil for easy flow as shown in Figure II.

Microimaging Rf birdcage coils and imaging

probes:

Microimaging probes or bird cage RF volume coil is

shown in Figure 1. The coil is attached with

physiological isofluorane anesthesia and vital

monitoring, 35 mm inner diameter of coil. In principle,

major goal of microimaging is to detect structural and

functional abnormalities of tissue.

Protein Targeting Assay and Gadolinium

Enhanced MRI:Theory

In MRI technique for protein targeting assay, proteinsubstrate is bound with ligand chelate and Gd by an

enzyme. In normal tissue, Gd contrast complex is

bioactivated and switched ON when enzyme cleaves

substrate from Gd3+ complex leaving Gd3+

accessible to water. In renal dysfunction, substrate

blocks the coordination site of Gd3+ due to enzyme

deficiency and inhibits access of water to chelated

Gd3+ ion (switch OFF). The difference in access of

water protons results to change in relaxivity (r1 or r2)

or change in the relaxation rate (1/T1 or 1/T2) s-1

per millimolar concentration of MRI contrast complex

normalized to the millimolar concentration of the Gdcontrast agent [10]. The change in relaxation times in

presence of bioactive enzyme protein reaction is the

basis of protein targeting assay or bioactive molecule

induced magnetization enhancement of MRI contrast

agent. The observed relaxation rate Tobs is given by

Eq 1.

1/Tobs = 1/T*+ ri [Gd]; i = 1, 2---n (1)

where T* is the relaxation time in the absence of a

contrast agent. Bioactive molecules are receptor,

enzyme, antibody, hormone etc. Plot of 1/ Tobs versus

Gd3+ concentration gives the r1 relaxivity as a slope

and r1 relaxivity expressed in mM-1sec-1 reflects the

ability of Gd3+ complex to increase relaxation. The

MRI signal intensity is proportional to 1/T1 or 1/T2 and

depends on both the concentration of the agent [Gd]

and magnetic resonance relaxivity. Amphiphilic

Gd(DTPA)(H2O) complex has l igand core

diethylene-triamine-penta-acetate(DTPA) attached

with Gd3+ (see Figure 3) by a biphenylcyclohexyl

moiety via a phosphodiester linkage top make

amphiphilic Gd complex. Gd complex targets serum

albumin, collagen, ferritin. Noncovalent binding of Gd

with protein restricts the rotation and distribution of

gadolinium only in blood vessels to make them

brighter on MRA due to slower rotational tumbling time

(longer R) with result of faster relaxation rates or

increased relaxivity or 1/Ti. As shown in Figure 3A,

seven unpaired electrons around GdIII create local

magnetic field. The R of Gd3+ is 50-100 picosecondswhile R of albumin is 50 ns. The protons of Gd bound

protein active groups precess at Larmor frequency

proportional to tumbling rate of gadolimium-protein

complex. In MRA, albumin binding with Gd slows

down tumbling rate of gadolinium complex and

increases proton relaxivity [protein induced

magnetization enhancement]. Noncovalent Gd3+

protein binding is major factor to delay the excretion of

gadolinium through kidneys or keeping gadolinium for

more time in circulation to enhance image contrast.

However, protein binding (protein bound fraction 65%)

has a pharmacodynamic diastereomer affinity effectKd depending on chain length in terms of increasing

the MR signal by increasing the relaxivity of the agent

by up to 10-fold but free unbound Gd3+ causes

toxicity. Other factors of magnetization enhancement

and increased relaxivity are applied magnetic field,

size of gadolinium complex, inner and second sphere

hydration states, rotational motion of complex, water

exchange rates, ion-proton distances and Gd3+

electron configuration. The measurement of Gd3+ and

ligand-protein interactions also explain the possibility

of functional in vivo MR imaging of renal and nephron

excretory function as following:1. The increased relaxivity and NMR relaxation

dispersion profi les were f irst described by

Solomon-Bloembergen-Morgan (SBM) for Gd-water

coordination assuming a pure state of Gd3+ with low

Zeeman energy and zero field splitting (D=0) while Gd

protein inner coordinat ion complexes of

Gd-BOPTA,Gd-EOB-DTPA showed D=0.02 - 0.06

cm-1 due to rotational motion and water exchange [10].

2. Gd-proton distance in different Gd-ligands as shown

in Figure 3A shows that 2.9 A vs 3.1 A makes more

50% difference of relaxivity ri in coordinated water

molecules. However, dipole interaction with Gd3+

electron spins (Gd-proton distance in parallel or



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perpendicular ligand structures) determined poor

anisotropic hyperfine interaction of proton inversely

proportional to relaxivity [10].

3. Protein hydration state controls the displacement of

water in inner or outer water spheres around protein

ligand described as basis of albumin carboxylated

chain targeted gadolinium complex [10].

r1obs = r1IS + r1SS + r1OS = q/[H2O]/T1m + m+

r1SS + r1OS (2)

where q is inner sphere hydration number and m is

life time of water molecules in Gd complex. Relaxivity

in inner sphere (IS), second sphere (SS) and outer

sphere (OS) are around complex.

4. At high magnetic field, gadolinium has strong effect

on T2 relaxation of proton due to Curie spin relaxation

dependence on slow water exchange and low m.

5. Protein binding with gadolinium determines therotational correlation time due to motional flexibility of

protein and rotation of coordinated water ligand about

Gd-O axis.

6. High magnetic field, gadolinium has strong effect on

T2 relaxation of proton due to Curie spin relaxation

dependent on water exchange and m.

7. Magnetization change in kidneys is measured as

arterial magnetization difference M at time t after

inversion pulse as:

M = 2Mo(F/p)te-t/T1 (3)

where F is blood flow in renal artery, p is ratio of

concentrations in renal tissue and artery, T is te-t/T1.8. Renal perfusion per unit volume (RP) is measured

as:

RP/Volume = Slope(max) / (1/T1)arterial (4)

Where slope(max) refers to the leading edge of the

first pass signal intensity vs time curve. The

denominator refers to the peak change in T1

relaxation rate in the arterial blood.

Image contrast is derived from T1 change; this

depends upon the product of relaxivity and the

paramagnetic ion concentration, (1/T1) = r1[Gd]. One

can increase the Gd payload as well as its relaxivity.

GFR can be estimated using T1 measurements beforeand after contrast administration in the renal artery

and vein as:

GFR = d[Gd]medulla/d[Gd]cortex = K.dt (5)

[Gd[artery vein]/Gdartery] RBF (1 Hct) (6)

where K is flow rate or renal clearance between two

compartments.

Large proteins ferritin, fibrin (constituent of blood clots)

and type I/III collagen (elevated in fibrosis) are other

contrast boosters in MRI [27]. Goal was to use

minimum sized complex with minimum rotational

internal motion to design gadolinium multimers. Dual

targeting or gadolinium DTPA linked with two ligands

target two peptide binding groups at C- or N-terminus

(high affinity site and hydrophobic aryl site of chelate

in balance) to keep minimum rotational freedom and

high relaxivity.

Nanoparticles as Imaging Contrast Agents:

After in vivo injection of GdIII Omniscan contrast agent

and iron-oxide magneto-ferritin SPIOM nanoparticles

in animals, both contrast agents accumulate in GBM of

rat kidney in glomerulus as a result of electrostatic

interaction of contrast agent with tissue proteins in

GBM regions of negative charge as shown in Figure

2B [26]. Other study showed that ferritin protein in

blood stream oxidizes and each ferritin molecule can

store around 1800-2500 iron atoms. Magnetoferritin

core acts as suitable in vivo delivery system of iron

oxide contrast agent [28]. Altered MRI signal

intensities at cationic magnetoferritin accumulatedsites can predict in vivo molecular nature and

dysfunction of renal vasculature as visible molecular

changes in GBM [28]. Distinct in vivo relaxivities and

susceptibility effects of SPIOM on MRI signal may be

described as following:

The nanoparticle SPIOM dephasing and MR signal

relationship can be shown as:

Signal = TE exp(-TE/T2*) (7)

where TE is echo delay time, T2* is transverse

relaxation constant due to susceptibility.

1/T2* = 1/T1 + 1/T2 (8)

where 1/T2* is dephasing signal due to SPIOMinduced myocard iac f iber spec i f ic f ie ld

inhomogeneities mesured by GEFC sequence. The

dephasing signal may be proportional to cubic

nanoparticle radius.

Cationic iron oxide from SPIOM gets attached with

renal basement membrane and subsequently taken up

by podocytes to accumulate in renal pelvis as

illustrated in Figure 1B[18,21-24]. Polyamidoamines

are unique dendrimers to visualize the vascular pools

in cortex and medulla to determine renal dysfunction

distinct from gadolinium dynamic contrast [23].

Moreover, SPIOM may be accumulated and bind withproteoglycans in heart, eye, muscle tissues [24].

Current view of gadolinium toxicity and nephrogenic

systemic fibrosis is not well understood. Present study

outlines the gadolinium induced nephrogenic systemic

fibrosis as a sequence of events due to protein

dysfunction in kidneys as shown in Figure 3. Present

study reports the physical nature of gadolinium toxicity

induced fibrosis in skin structures and renal

dysfunction similar with NSF as public concern in

support of extra care to use gadolinium based contrast

agents in clinical imaging. The purpose of study was to

compare the Gd contrast enhancement with contrast

enhancement method by SPIOM nanoparticles to



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explore new alternative with less risk of renal toxicity

and fibrosis by measurement of skin and renal micro

structures at 900 MHz as MR microimaging

biomarkers. Novelty of the study is:

1. Clear evidence of increased skin epidermis

thickness, hair follicle size associated with NSF;

2. Gd-protein binding assay development;

3. New approach of contrast enhancement by SPIOM;

4. first time micro details of skin and SPIOM based

kidney structures at 900 MHz NMR apparatus.

Implications are: safe and restricted use of Gd; better

Gd vs SPIOM information as guide to preclinicalstudy;

possible use of SPIOM in evaluation of dynamic renal

injury.

Materials and methods

Rat animals and imaging contrast agents:

Three-week-old male Spring Dawley rats weighing

100125 g were maintained five animals per plastic

cage and used for MRI experiments in compliance

with the Animal Care Ethics Committee guidelines and

animal ICUC protocol [25]. Locally raised rat animals

were purchased from Pet center, Monroe street,

Tallahassee. Cationized magnetoferritin (SPIOM) was

a gift from center of nanomagnetics and biotechnology,

Florida State University and Sigma Aldrich (St Louis,

MO). Omniscan (GE Healthcare) were obtained as giftfrom Dr Chales WIlliam at Tallahassee Memorial

Hospital. Gadodiamide was used as Omniscan

(without 5% caldiamide) with calcium ions bound to

caldiamide and Gd ions.

Ferritin binding assay:Noncovalent protein binding

assay method was developed for titrating interaction

between Gd3+and ferritin to demonstrate the extent of

relaxation enhancement E. The water proton T1

relaxation constants [1/T1obs]Gd-ferritin of 0.1 mM

solution of Gd3+ with different concentrations of ferritin

(100-700 g/mL) were measured at 20 MHz, 37C

keeping relaxation constant [1/T1obs]Gd at same

concentration of Gd.

Gd + Ferritin ----> Gd-Ferritin

E = [1/T1obs]Gd-ferritin / [1/T1obs]Gd (9)

where 1/T1obs is [1/T1obs]Gd-ferritin + [1/T1obs]Gd

1000

The effect of gadolinium exposure at different time

intervals on skin was established due to gadolinium

induced changes in epidermis thickness and hair

follicle size as shown in Figure III.

SPIOM contrast agent assay:The dephasing effect

by T2*

was measured at different concentrations(100-700 g/mL) of 0.1 mM solution of SPIOM

contrast agent. The change in relaxivity was observed

at 20 MHz, 37 C as expressed in Equations 7 and 8.

In vivo MRI at 500 MHz:

Rat animal preparation: Five rat animals were

anesthetized by intubating with 14 gauge, 2 inch

intravenous catheter (Abbocath Lab, IL) on nose with30% oxygen/70% nitrogen mixture containing 5%

isoflurane/air mixture to continuous supply through

nose during MRI session.

Animals were kept in vertical direction to the side of

MRI gantry and 30 mm diameter RF insert covering

kidneys in the center of magnet. Multislice gradient

echo images were acquired with 30 flip angle and

TE/TR 5/25 ms. The resolution was 100 100 500

microns with 256 256 matrix; NEX= 2. Two methods

were used in MRI to enhance the tissue contrast: 1.

Using alternate nanoparticle based MRI; 2.

Gadolinium enhanced MRI. Simultaneously animals in

one set were given injection of ferritin bound SPIOM in

physiological saline (3.0-3.3 mg/100 mg weight) in one

shot of optimized period over 1 minute for 1 hour

experiment [28]. After in vivo experiment was over in

1-1.5 hour to acquire contrast enhanced in vivo

images, each animal was sacrificed by perfusion with

saline and 4% paraformaldehyde over 3 minutes.

Other set of animals in same conditions were treated

with multiple (three times in one week with 48 hour

intervals) intravenous injections of Omniscan

(gadodiamide; Gd-DTPA-BMA) at the dose of 2.5

mmol Gd/kg body weight equivalent to clinical dose.

After 24 hours of last injection of SPIOM or gadolinium

treatment or perfusion for in vivo MR imaging, animals

were sacrificed for ex vivo 900 MHz MRI experiments.

After sacrifice of animals, kidneys were removed,

dissected out from adipose tissue and placed in 2%

glutaraldehyde in PBS buffer pH at 7.4. For gross skin

changes, abdomen of animal was shaved for better

visibility. Skin specimens were excised and fixed in 4%

neutral buffered formalin. After routine dehydration, all

tissue samples were embedded in paraffin and

sectioned (5 micron) for hematoxylin and eosinstaining [26]. Axial T1-weighted gradient echo

sequence for dynamic imaging (TR=130 ms, TE=1.0

ms, flip angle 90) was done after 30 ml intravenous

gadolinium contrast injection for acquiring precontrast

and post-contrast images in arterial and nephrogenic

phase to distinguish lesion. Coronal 3D fast gradient

echo with fat suppression was used for delayed

contrast-enhanced images (TR=3 ms, TE=2 ms, flip

angle=15o) was used to evaluate solid tissue in

perinephric fat. Two methods were used in renal MRI

to enhance the contrast: 1. Using alternate

nanoparticle based MRI; 2. Gadolinium enhanced MRI.On images, points on the grid overlying the kidney



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were counted as locations of cortex (PC), medulla

(PM), or renal pelvis (PP) (see Figure 2B). The cortex

was defined as the area superficial to the arcuate

arteries in dynamic MRI images. Kidney volume (VKid)

was estimated as sum area of each microstructure [

a(p) = a(p)cortex+ medulla+pelvis] by following

formula

VKid = 10 PS[a(p)]T (10)

The cortex-to-medulla ratio (VC/M) was estimated

using the formula

mVC/M = (10) PC [a(p)T]/10] PM a(p) T (11)

where 10 is the reciprocal of the sampling fraction, Ps

is the total number of points counted (sum of PC, PM,

PP), a(p) is the area associated with each grid point,

and T is the section thickness. Nephron number and

glomerular volume. Glomerular number and volume

were determined using unbiased stereologicalmethods as previously described [29]. The total

number of glomeruli in a kidney was estimated using a

physical dissector/fractionator combination [29]. A 2

2-cm grid was placed over each field of view, and

points falling on kidney tissue (PKid), glomeruli

(PGlom), and renal corpuscles (PCorp; the filtration

unit of the kidney made up of Bowmans capsule and

the glomerulus) were counted. Glomeruli sampled by

an unbiased counting frame on the field of view on the

tenth section that were not present in the eleventh

section were counted. Glomeruli sampled in the

eleventh section that were not present in the tenthsection were counted to double the efficiency of the

technique. This process was repeated for each

complete pair of sections. The number of section pairs

used for counting glomeruli in each kidney. Total

nephron number (NGlom, Kid) was then estimated

using the following formula:

NGlom, Kid (10 ) PS/PF (1/2fa)Q (12)

where 10 is the reciprocal of the sampling fraction, PS

is the number of points overlying all kidney sections,

PF is the number of points overlying complete kidney

sections, 12fa is the fraction of the total section area

used to count glomeruli, and Q is the actual number ofglomeruli counted. The glomerulus was defined as the

glomerular tuft. Mean glomerular tuft volume (VGlom)

was estimated using the following formula

VGlom = VV(Glom, Kid)/NV(Glom, Kid) (13)

where VV(Glom, Kid) is the volume density of

glomeruli in the kidney and was estimated by dividing

PCorp by PKid. NV(Glom, Kid) was calculated by

dividing NGlom, Kid by VKid.

Ex vivo MRI images and corresponding histology

images were compared for the measured size of each

visible structure in skin and kidneys.

NMR spectroscopy and Histology

After in vivo 500 MHz MRI imaging of animal and ex

vivo 900 MHz MRI imaging, excised skin specimens

from abdominal region and whole kidneys were

excised processed for proton NMR spectroscopy. After

NMR/MRI experiments, each excised skin and kidney

tissue was perfused with saline and fomblin solution

and stored in 30 % sucrose solution for histopathology.

For detail microscopy, skin and kidney tissues were

processed in 10% neutral buffered formalin solution

and dehydrated through a graded series of alcohol

treatment, embedded in paraffin, and cut into 4 micron

sagittal sections (Histoserve, Germantown,MD) and

stained with hematoxylineosin stain [31]. The histology

sections were rinsed twice with phosphate buffered

saline PBS pH 7.4 and examined by high field

microscopy and digital images were captured for

morphology [26].

Proton quantitative NMR spectroscopyAll water-soluble perchloric acid extracts and lipid

extracts from excised skin and kidney tissues were

analyzed using a 500 MHz high resolution Bruker DRX

system (Bruker Biospin, Inc., Fremont, CA, USA) [31].

An inverse TXI 5-mm probehead was used for all

experiments. In order to suppress water residue in

extracts, a standard Bruker water presaturation

sequence was used at operating frequency for proton

channel: 500 MHz; power level p11=3 dB; power level

for water suppression p12=55 dB; power angle p1=7.5

sec (90opulse); power angle for water suppression

p12 = 60 ls; water suppression at O1 = 4.76 ppm;relaxation delay d1 = 12.85 sec (5T1); delay for

power switching d12 = 20 ls; short delay d13 = 3 ls;

spectral width sw = 12 ppm; total number of scans ns

= 40. An external standard substance, trimethylsilyl

propionic- 2,2,3,3,-d4 acid (TMSP, 20 and 50 mmol/L

in D2O) was added into a thin glass capillary. The final

TMSP concentration (0.5 mmol/L and 1.2 mmol/L) in

the capillary was calculated prior to NMR experiments

on study extracts using a standard amino acid solution.

The TMSP capillary was placed into the NMR tube

during the experiment (0.5 mmol/L for water-soluble

extracts and 1.2 mmol/L for lipid extracts), and served

as an external standard which allowed for absolute

metabolite quantification in each study extract. 1H

chemical shifts were referred to TMSP signal at 0 ppm.

After performing Fourier transformation and making

phase and baseline corrections, each 1H peak was

integrated using 1D XWIN-NMR program (Bruker

Biospin, Inc., Fremont, CA, USA). The absolute

concentrations of single metabolites were then

referred to the TMSP integral and calculated according

to equation 11:

Cx = Ix : Nx CI : 9 V : M (11)

where Cx = metabolite concentration, Ix = integral of

metabolite 1H peak, Nx = number of protons in



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metabolite 1H peak (from CH, CH2, CH3), C = TMSP

concentration, I = integral of TMSP 1H peak at 0 ppm

(for TMSP 9 protons), V = volume of the extract, M =

weight of kidney tissue or volume of blood sample.

Briefly, skin and kidney tissues (10-15 g wt) were

treated with 4% perchloric acid and extracts were

analyzed for small metabolites by NMR spectroscopy

as described in previous study [32]. In order to confirm

the identified structures HSQC was run on standard

solutions of 10 mmol/L allantoin (Sigma Aldrich, St.

Louis, MO, USA). The ratios of dominating spectral

peaks were calculated as metabolomics molecular

signatures of fibrosis in excised skin and kidney.

Data processing:Image reconstruction was done in

Paravision (Bruker Biospin, Billerica, MA). MR images

were analyzed using Bruker Paravision with GIMP

software and measurements were made by Image J(national Institute of Health, Bethesda, MD) package

[17]. Receiver gains on MRI system were adjusted

between imaging sessions. Image contrast and

brightness were adjusted to high light contrast

between skin or kidney tissue and other surrounding

tissue. Statistical analysis: Gadolinium enhanced

tissue measurements were evaluated for skin and

kidneys tested globally for differences in medians

between 2 groups with nonparametric Kruskal-Wallis

test (Dunn test). Pairwise comparison was done in

case of significance at a twosided 5% level of

significance [26].

Results

The titration plot in Figure 5A showed enhancement

factor or ratio of paramagnetic longitudinal relaxation

rates (1/T1)para [s-1] in absence and in presence of

different increasing ferritin concentrations at constant

Gd3+ concentration 0.1 mM. The 1/T1 [s-1] increase

of Gd-ferritin solutions (in presence of different

concentrations of ferritin) was much larger than

solution of Gd3+ alone but did not increase linearlywith ferritin concentrations. Increased enhancement

factor E was 4.5 fold suggested the ferritin binding with

Gd3+ consider ing 0.125 mM Gd3+ actual

concentration in solution. The measured r1 or 1/T1

includes three components as shown in Equation 9.

SPIOM nanoparticles had average radius 15-25 nm.

The contrast enhancement was based on

dephasing or darkening on T2 images. The SPIOM

iron oxide particles showed dephasing or negative

contrast of accumulated particles. The MRI signal

intensity changed with concentration of SPIOM as

shown Figures 5A and 5B. Gadolinium treated ratsshowed skin reddening, fur loss, scab formation and

ulceration. SPIOM treated rats did not show any

visible change. At 500 MHz, rats were imaged in vivo

at 500 MHz for gross skin T2 weighted images. Rat

animal images before and after SPIOM showed dark

regions of epidermis and hair follicles. Pre- and

post-gadolinium treatment showed distinct skin

structures with bright stratum corneum, adipose tissue

and sebaceous gland as shown in Figure 6. The

epidermis, dermis, hair follicle were major MRI visible

structures in skin. Gadolinium enhanced the contrast

of skin structures hair, epidermis, dermis, hair follicle.

Gadolinium overdose caused clefts with fibrosis in

dermis, exfoliated epidermal thickening and infiltration

of dermis as distinct features as shown in Figure VI.

Rat in vivo MRI images of kidneys showed distinct

cortex and medulla regions. The ureter and pelvis

were distinct and brighter on post Gd enhancedimages. Rat T2 weighted images before and after

SPIOM showed darker medulla, pyramid, distinct from

cortex, ureter. The darkness may be corresponding

with flowing presence of nanoparticles in vasculature.

Pre-gadolinium and post-gadolinium treated animals

showed measurable distinct structures in both skin

and kidneys. Major changes were: sebaceous gland

increased size, hair follicle thickness, epidermis

thickness in skin and increased pelvis size, cortex size

in kidney (see panel B in Figure 6).

Pre-SPIOM and post-SPIOM treated animals showed

measurable better contrast of structures in both skinand kidneys. Major changes were: bright stratum

corneum, gray epidermis and hypointense dermis with

brighter vasculature, darker hair, brighter pelvis,

grayish pyramid, brighter cortex, isointense medulla

(see panel C in Figure 6). Calculated renal volume

(Vkid = (lhw) 2/3) was 0.9-1.2 cc; calculated

V(cortex / medulla) ratio was 1.2-1.5 by MRI using grid

method (see Figure 6). The comparison showed 20%

less measured Vkid and V(cortex/medulla) by

histology than measured by MRI. Nephron number in

cortex was measured 850-1300 per kidney by

histology under high power microscopy but notconsistent. The coefficient of variation was 2.3%,

when nephron number was counted three times in one

excised kidney.

After 500 MHz MRI imaging, excised skin samples and

kidneys showed distinct structurtes by 900 MHz

microimaging and histology. Major observations were;

1. ex vivo skin epidermis and hair follicle changes;

2. ex vivo gradient-recalled echo(GRE) MRM images

of kidneys showed distinct renal cortex with

magnifications;

3. images showed distinct spots of low signal intensity

in cortex in injected animals;

4. location, size and density of dark spots after



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dephasing were consistent with uniform and specific

glomeruli in cortex visible after injection of SPIOM;

5. renal pelvis was visible with accumulated SPIOM.

The dark changes due to gadolinium and SPIOM

contrast changes in MRI signal of skin are shown in

Figure V and kidney MRI signal in Figure

6. Thickening of epidermis in skin: The rat skin hair

follicle and epidermis areas were distinct on T2

weighted images comparable with histology digital

images showing sensitivity 77% specificity 11%,

accuracy 83% and precision 66.6% similar to our

previous report [26]. MR microimaging of excised skin

tissue measured epidermis 100 m, dermis 600 m,

hair follicle 50-70 m, sebaceous gland 50-60 m.

Gadolinium treated skin epidermis measured 200 m

or more, dermis 750-800 m rich in collagen as shown

in Figure VII (panel A). Hair body measured 500 mlong with 100 m thick hair follicle. Gadolinium treated

skin showed epidermis thickening 2-5 times over

normal thickness and hair follicle changed 2-3 times

with sebaceous gland enlargement as shown in Figure

7 (panel B). Skin excised from SPIOM injected rat

showed accumulation of particles with severe

dephased signal of distinct epidermis and distinct hair

body with measurable hair follicle appeared darker as

shown in Figure 7 (panel C).

Disrupted renal basement membrane: The basement

membrane of kidney was visible on histology as

shown in Figure 2B while it was not visible on 21 TeslaMRI images.

MRI signal intensity profile: The signal intensity profile

of a line passing epidermis, dermis and hair follicle in

skin tissue showed distinct line intensities of each skin

structure. The signal intensity profile of a line passing

renal cortex and power spectrum showed highest

contrast between cortex and medulla and poor

contrast between renal pelvis and ureter in kidney.

The line profile showed less variation in signal

intensity corresponding to a peak at 1.25/mm on graph

or 500 m distance in two points on x axis as dark

spots shown in Figure VIII. The accumulation ofSPIOM in kidney cortex was detected with MRI in vivo

after 5 intravenous injections. Kidney slices in 100 x

500 m are shown showing distinct cortex as dark

spots. The medulla and pelvis were less visible. The

signal intensity normalized with mean intensity on

average spatial power spectrum (n = 10 lines) showed

line profile oscillation 1.50 /mm or 1 spot per 500 m)

while at 900 MHz half of the frequency determined ex

vivo as shown in Figure 8. It indicated the partial

volume effects between glomerulus with low resolution

in vivo image resolution. Ex vivo 900 MHz MRM

isotropic images showed resolution 1 mm3 by GRE

MRI imaging of normal rat kidney. The gadolinium

treated normal kidneys showed hypointense spots in

cortex as shown in Figure 6. Vascular dynamic

information was absent but distinct dark and white

bands in cortex and subcapsular medulla. With

increased gadolinium exposure time, dark spots

showed hypointense without contrast between

glomeruli and cortex. These images indicated

sclerosis or fibrosis (wider pyramid, large pelvis area,

enlarged cortex region in some parts of cortex) with

glomerular damage (NSF) with loss of glomerular

integrity (fibrosis or overburdened glomerulus

manifestation of slow permeation of large proteins

through glomerulus and slow uptake of filtered

proteins by proximal tubule). Ex vivo renal cortex

signal intensity, normalized with surrounding tissue

was distinct. The dephased MRI signal was lower in

SPIOM injected normal cortex. It indicated the kidneyprotein excretory ability due to breakdown of

basement membrane. Histology showed skin

minimal-to-moderate increased cellularity (fibroblast

cells, dendritic cells with lymphocytes) and fibrosis in

dermis resembling NSF after Omniscan treatment.

The dermatopathy by post Gd treatment was distinct

by histology as shown in Figure 7 in insert. Renal

morphologic alterations due to gadolinium exposure

were visible on histology due to basement membrane

injury, cell death, fibrosis or sclerosis in glomeruli.

Comparison of MRI and histology digital images

showed very distinct areas as bands:1.outermost cortex bright band;

2.dark band of deep cortical region;

3.bright band in outer stripe of outer medulla showing

projections into deep cortical region rich in proximal

tubules (appear as rays);

4.dark band in outer half of inner stripe of outer

medulla; and

5.a bright outline of pyramid and ureter space (renal

fornices). It was poorly distinct on in vivo 65 m (at

500 MHz) and better at ex vivo 900 MHz 15 m

isotropic kidney images. Comparison of post-Gd MRI

images with histology digital images showed twodistinct structures:

1. middle white band of outer medulla (outer stripe rich

n proximal tubules) enlarged in GdIII treated kidney;

2.black band in deep cortex (inner stripe) unchanged,

as shown in Figure 9 (middle panels A and B). Fibrosis

of nephron tubule was localized and distinct as shown

in Figure 9 (panels at the bottom). Gadolinium treated

kidneys showed good contrast of bright band of outer

stripe and diminished superficial cortex with delayed

enhancement of inner medulla with better details of

subcapsular stripe as distinct structures than the

dephased contrast by accumulation of SPIOM. The

background signal intensity was hypointense or darker



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matched with histology findings of increased vesicles

in the fused podocyte foot processes and endothelia

detached from basement membrane in the glomeruli.

Gadolinium also gets accumulated in urinary space

and podocyte cell body due to possible gadolinium

toxicity seen as swollen or bizarre pelvis on histology.

So, SPIOM distribution from basement membrane to

cell body after basement membrane breakdown may

also cause SPIOM accumulation in cortex that

generates dark signal due to dephasing. The

histological changes as accumulated SPIOM are

shown in Figure 9 (see panels at the bottom).

Measurement of skin, kidney structures by gadolinium

vs SPIOM: The epidermis thickening in pre-Gd

specimen at baseline was minimum 0.13 mm and

increased significantly up to 0.36 mm. On average,

33-36% epidermis thickening for epidermis, 10-15%dermis, hair follicle 40- 45% and sebaceous gland

30-40% enlargement was measured at follow up

exposure up to 6 h post-Gd treatment. Renal regions

were measured as: whole kidney 4.5-5 mm; cortex

600 m; medulla 400 m; pelvis region 2.5-3.0 mm at

the middle of kidney in axial plane. The measurement

by histology was 20% less than the measurement by

MRI method. Comaprison of Gd treated and SPIOM

treated kidney specimens showed distinct medulla

regions as enhanced bright by Gd treatment while

dark by SPIOM treatment. The major difference was

the visible ray like appearance of proximal tubule richregions got enhanced with gadolinium treatment. On

histology, both Gd treated nephrosis and accumulated

SPIOM sites were distinct as shown in Table 2 and

Figure 9 (panels at the bottom). Evaluation of skin

dermatopathy and renal fibrosis: The post-Gd skin

MRI images and histology showed visible changes of

clefts, ulceration in epidermis, widening of hair follicle

size (x2), brighter oil glands (+30%), dermal fibrosis,

accumulated contrast agent (-80% of area),

acanthosis. The kidney specimen showed visible

changes of wide pelvis, subcapsular band wide,

hypointense medulla (-40%), swelling of pyramids(+1.5-2.0 fold). MRI specific measurements were: loss

of bright cortex region, wide and thickened white band

of proximal tubule rich inner cortex-outer medulla

layers (x 3) and missed subcapsular rays in post-Gd

kidney and dark medulla and pelvis with accumulated

particles covering more than two-third kidney area. In

high power field microscopy showed distinct

cytomorphology of fibrosis in skin epidermis and renal

nephron cells as shown in Table 3 and Figure 9. NMR

spectroscopy and fibrosis: The excised renal extracts

showed a distinct spectra of small molecules including

amino acids, monosaccharide and urea cycle products

(see Table 4). Amino acids in skin were earlier

reported[26]. Three NMR peak assignments at 5.25,

5.39, and 5.45 ppm position were identified as a

glucose, (CH) group of allantoin, and glycogen (see

inserts in Figure 10). Allantoin was assigned as the

end product of xanthine metabolism and uric acid.

Other major NMR peaks were triglyceride, cholesterol,

PUFA, glucose, glycogen as markers of oxidative

stress.

Discussion

The multiple gadolinium injection schemes in

prolonged systemic exposure of animal to Omniscan

contrast agent resembled with human patients with

severe renal impaired function after triple dose of

contrast agent. The present study reflects the

macroscopic and microscopic skin changes

associated with histopathologic characteristics similar

to findings in human NSF patients with better

information of dermal fibrosis (macroscopic skin

lesions, increased dermal dendritic cells and spindle

shaped fibrocytes). Earlier study described the role of

panniculus carnosus in dermal fibrosis in rat skin [33].

Rat studies often achieve a higher signal-to-noise ratio

(SNR) and offer more robust and better controllable

physiological conditions under anesthesia. Glomerular

filtration rate measurement is current state of art. It is

measured by three methods:1. GFR measurement as linear relationship of 1/T1 to

the serial sampling of serum and urine [34]; 2. Using

extraction fraction of Gd-DTPA, GFR is calculated as

(Gdartery [Gdvein/Gdartery]). The gadolinium

concentrations can be calculated by measuring T1

constants of ar tery and venous blood in

pre-gadolinium and post gadolinium administration as:

1/T1post = 1/T1pre + [Gd] x R,

where R is relxivity of gadolinium. The GFR =

EFRBF(1 Hct), where RBF is renal blood flow

measured by phase contrast flow quantification and

Hct is measured by hematocrit.[35]; 3. Using contrastenhanced dynamic MRI based on time dependent

gadolinium DTPA in cortex and medulla morphometry

was performed [36]. Dynamic study on renal function

and GFR by MRI was not evaluated due to limitations

of animal conditions such as SAR, exposure of radio

frequency and high magnetic field exposure. Gd3+

complex exhibit bioactive protein molecule induced

magnetization enhancement (or increased relaxivity)

based protein or enzyme activated MRI requires

overdose of Gd contrast agent to keep optimum

available free Gd concentration (0.1 mM free Gd

needs dose of 0.125 mM contrast agent) to generatesufficient MRI visible signal intensity. Perhaps, extra



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dose of 0.025 mM Gd3+may cause NSF. Gadolinium

based nanoparticle targeted applications were

reported in MRI for surface molecule/second molecule

binding (ligand/receptor), complementary nucleic acids,

avidin/biotin [37], amyloid beta1-40 peptide/amyloid

beta [38], antibody/antigen [39]. Gadolinium oxide

nanoparticles have emerged as multimodal contrast

agents [40]. The present study indicates the growing

application of enzyme activated MRI contrast agents

as enzyme relaxivity signatures of tissue gene

expression with enzyme activity distributed in tissues

and cells.

Comparison of SPIOM vs Gadolinium treatment:

SPIOM toxicity is not known on skin or kidney tissues

but it well tolerated in tumors to perform microimaging

to determine vasculature and iron accumulated sites of

injury in tissue[1,25]. Gadolinium is primarily used asfiltration marker of dynamic renal function or GFR,

nephron tube functionality. Gadolinium toxicity is

documented as causing NSF [1,2]. Gadolinium toxicity

is attributed due to targeting tissue proteins (skin

collagen I/III bundles and nephron basement collagen

type 1/2 IV proteins) [18-21,25]. The effect of

delayed gadolinium treatment on MR signal intensity is

to improve tissue contrast by shortening of T2 or T1

constants [21,22]. Irony is that gadolinium based MRI

contrast agents were reported as best pH sensors in

tissues and enzyme activated MRI agents for

magnetization enhancement of tissue [41,42]. Theeffect of SPIOM is to diphase the MR signal and

enlarge or delayed growth of T2 or T1 relaxation

constants to generate darkness around the

accumulated SPIOM contrast agent molecules. New

developments are anticipated in the T2 weighted

diffusion weighted MR imaging. The outcome depends

on MR signal influenced by Brownian motion of water

molecules such as restricted motion of water

molecules across cell membrane, viscosity. However,

high T2 signal of restricted water is not understood

[43]. Alternatively, ADC maps from two images at

different gradient duration and amplitude (b-values)

showed difference between hydronephrosis and

pyelonephrosis; pyelocalcyceal system as hypointense

in hydronephrosis and hyperintense in pyonephrosis

[44]. Further work is expected to evaluate toxicity of

SPIOM used as imaging contrasts at optimal

concentration delivered. However, some diseased

conditions such as chronic transfusion, anemia

syndromes may not be delivered optimal required

dose [29]. The magnetoferritin may be good to use

less iron dose while generating increased relaxivity

signal. On other hand, magnetoferritin or apoferritin

may not be the good choice if it is loaded with

gadolinium because of gadolinium chelates causing

NSF [45-46]. Gadolinium chelates filled with ferritin

have been reported in tumor in vivo with gadolinium

bolus of 0.01 mmol [47]. SPIOM acts negative MRI

contrast agent in enhancing the dark contrast on

T2-weighted MR images than the surrounding regions.

Dark spots correspond to microspheres existing in

blood vessels.

Fibrosis and Gd toxicity:

Epidermis thickening was not clearly visible on in vivo

MR images with no visible evidence of any effect of

gadolinium or SPIOM. High field 900 MHz MRM

microimaging showed distinct epidermis thickening

after the Omniscangadolinium skin exposure. Earlier

study showed accumulation of gadolinium in skin 1.7

0.2 mol/g with severe NSF-like skin lesions [1].

Major improvements were: (1) the high contrast of the

SPIOM based reagent permitted us to reduce the slicethickness from 5 mm (in previous rabbit studies) to 0.8

mm; (2) use of a bird cage-type coil allowed uniform

imaging of both kidneys at a voxel size of 160 160

800 mm3(0.8 mm thick slice); and (3) improvements in

MR instrumentation allowed the image matrix to be

decreased from 0.62 mm to 0.16 mm. Skin fibrosis

with sclerosis of renal structures were associated with

burden on glomerulus to excrete out at normal rate

that keeps nephrons with optimal pressure gradient

across basement membrane in nephron tube to

absorb/filter solutes/protein. Basement membrane play

significant role in different/absorption process at thecost of active energy in active filtration against

pressure gradient. Omniscan with excessive

gadolinium and caldiamide l igand and low

thermodynamic stability was reported to have NSFlike

skin lesions independent of ligand amount but

dependent on stability of Gd-chelates or dissociation

of Gd-complexes [1]. The present study showed

possibility of qualitative but unconfirmed correlation

between onset of NSF-like pathologic signs and

release of Gd3+ions in skin and kidney.

High field MR microimaging increased SNR with high

resolution or fast imaging. High field MR imaging can

be combined with SENSE or parallel imaging [16]. It is

attributed that overburden of gadolinium or SPIOM or

basement membrane disturbs the process of active

filtration (saturating basement proteins or exceeding

the maximum efficiency of nephrone tube or GFR)[25].

In the process of ATP dependent ATPase delayed

action or creatine clearance, nephrone tube get

exhausted or cause resistence or fibrosis or sclerosis

or swollen or increased size of pelvis, cortex. In 500

MHz MRM showed limited visibility of renal structures

and skin structures.

Fibrosis and microimaging:



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Epidermis thickening, dermis collagen I/III bundle

uncoiling has been reported with skin reddening,

reduced skin tensile strength, skin tissue flexibility [18,

45]. Gadolinium exposed 6-8 hours skin MR images

were reported with wider hair follicles (raised plaques

or papules), altered skin pigmentation on surface and

mucin [1]. The skin changes were attributed by effect

of interaction between gadolinium-ligands or chelates

with skin tissue proteins. Present study reports the

possibility of gadolinium toxicity as a result of

interactions between gadolinium and ligands plus

interaction between gadolinium and tissue proteins

(collagen I/III bundles in skin and collagen 1/2 IV rich

basement proteins in kidney). Information on

interaction of gadolinium with proteins in nephrons is

very limited [1,10-14]. Possible proteins are collagen

I/III, ferritin, TGF , Transglutaminase in skin targetedby gadolinium imaging contrast agents. Cationic

superparamagnetic iron oxide (SPIOM; Fe3O

magnetitie) may have interaction with tissue proteins

[23,25,48]. Our previous report suggested the

interaction with SPIOM ferritin with myoglobin in

flowing blood and cardiac muscles [49]. It is possible

that SPIOM is bound with skin collagen I/III proteins

but accumulation of contrast agents is not visible due

to poor vasculature in comparison with nephron tissue.

SPIOM was distinct in renal tissues because of high

vasculature. Cationic superparamagnetic iron oxide

(SPIOM; Fe3O4 magnetitie) is biocompatible andbiodegradable with promise of applications in animal

imaging. The MR relaxivity of magnetoferritin is low

2-10 mM-1sec-1. The cation iron oxide magnetoferritin

can be filled up with gadolinium chelates and forms a

potent MRI contrast agents to target basement

membrane [45,46]. The SPIOM may accumulate or

stay longer in blood pool or vasculature in kidney if

there is loss of high negatively charge of

proteoglycans in basement membrane [1]. The

accumulated nanoparticle sites visualized (dark

hypointense region) the basement membrane

breakdown down to the below 15 micrometers scaleusing 900 MHz MR microscopy as first demonstration

of targeting injected cationic iron oxide to the

basement membrane. Recently, use of ultra small

superparamagnetic iron oxide particles (USPIO) as

negative contrast agent was reported to have iron

oxide core covered with a low molecular weight

dextran coating [50]. USPIO get ingested by

macrophages through phagocytosis to cause loss of

signal intensity on T2- and T2*-weighted images.

Gradient echo sequences are the most sensitive for

these susceptibility effects. Technique is improving to

detect renal morphometric in vivo details very useful to

scientists to use nephron sparing microscopy. MRI

shows a moderate to high sensitivity in detecting

pseudocapsule as a hypotense rim on both

T1-weighted and T2-weighted images on gadolinium

enhanced GRE images.[51] In present prospective

study using quantitative metabolomics of kidney may

be useful for transplant patients with elevated serum

Cr levels who would undergo blood sampling for

assessment of their metabolic profiles. These

metabolic fingerprints could then be correlated with

the histologic findings previously if obtained by renal

biopsy. One could then determine early metabolic

profiles to predict and to differentiate between normal

form IR injury, rejection, and drug toxicity.

Clinical implications: Gadolinium related

nephrogenic fibrosing dermatopathy was reported with

skin symptoms including iching, thickening or swelling

of skin (shiny thick and hard skin), dark patches.Nephrogenic systemic fibrosis was associated with

MR urography visualizing pyelocalyceal systems and

ureters using heavily T2-weighted images or

T1-weighted images with gadolinium contrast. Heavy

T2-weighted images show urine in pyelocalyceal

system and ureters as brighter because of T2

relaxation time. The HASTE or single shot fast spin

echo (SSFSE) is suitable as fast with sufficient

in-plane resolution [36]. At high magnetic field it is

easy to get thin slices and maximum intensity

projection to overview the tract. For T2 weighted MR

urography. The use of relaxation enhancement (RARE)urograms and gadolinium enhanced 3D fast low angle

shot (FLASH) further enhance the signal. Intravenous

gadolinium combined with T1 weighted 3D gradient

echo sequence or fast 3D GRE EPI sequence can be

used to reduce ghost artifacts caused by peristalsis

[52]. GRE images provide better high resolution

images better than EPI images. Gadolinium is

excreted and get concentrated to show up as T2 *

signal loss. MRI has better information to detect

edema in kidney [53]. In MR urography, calculus

shows nonspecific signal void, blood clots, papilla, as

hypointense within bright urine. These NSF related

changes were related with transglutaminase

overactivity after at least 7 days of gadolinium injection

and transglutaminase inhibitors were recommended to

prevent the reoccurrences of NSF [54]

Limitations of gadolinium MRI micro imaging:

Gadolinium concentration released and amount of

bound ligand in gadolinium chelated imaging contrast

agents differ and both affect distribution kinetics,

thermodynamic stability. Renal function tests do not

provide information of each kidney while MRI gives

both functional and anatomic details with physiologicinformation. Slope of gadolinium DTPA enhancement



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in relation to gadolinium concentration in aorta is used

to calculate renal blood flow by pre-contrast and

post-contrast difference in relaxation constants. The

gadolinium concentration in relation with the difference

in relaxation constants of gadolinium phantoms can

calibrate the linear relationship of gadolinium

concentration with MRI signal intensity. In addition,

calculation of glomerular filtration rate is important

kidney function that needs attention after gadolinium

treatment and NSF. Still the skin and renal

complications after NSF caused by Gadolinium are not

completely known. An attempt is made to establish

physical basis of Gadolinium toxicity to result NFD and

NSF. In this regard other concerns of cardiovascular

safety, hemodialysis, calciphylaxis with metastasis,

renal insufficiency are also important as a result of

NFD and NSF in relation with gadolinium toxicity[55].Alternate gadolinium alumoxane and oxide

compounds are future candidates and will replace the

cyclic chelate ligands [56]. Possibly, limitations of renal

diffusion will be lesser to measure dynamic renal

function in prospective kidney donors in renal

transplantation [57, 58].

Conclusion(s)

The type of ligand in gadolinium chelates bound with

tissue proteins may play role to cause gadoliniumtoxicity resembling with NSF. MRI visualizes skin and

renal tissues with oil or fat gadolinium toxicity.

Structural MRI of skin and functional MRI of kidney

may have increasing role to evaluate gadolinium

toxicity. A SPIOM nanoparticle serve as novel

MRI-detectable vasculature as negative MR image

contrast on noninvasive MR detection of basement

membrane. The SPIOM particles injected into blood

vessel target proteoglycans in kidneys in vivo and

appear as dark spots in blood vessels and renal cortex

but not visible in skin.glands or intracellular fat. MRI is

highly sensitive to skin epidermis and hair follicle tomeasure.

Acknowledgement(s)

Author acknowledges the supply of nanoparticles from

Professor C.J.Chen at Center of Nanomagnetics and

Biotechnology and chemical-biomedical engineering

for departmental research funds and ACUC protocol.

The author also acknowledges the assistance of Dr.

Bruce R. Locke for his generous help in supporting in

vivo 500 MHz animal skin experiments andsuggestions. The assistance of Dr. Suniket Fulzele,

Kiran Shetty, Ashley Blue and Dr. William Brey from

the National High Magnetic Field Laboratory,

Tallahassee, FL, in standardizing experiments is

acknowledged.

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Illustrations

Illustration 1

Table 1



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Illustration 2

Table 2



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Illustration 3

Table 3



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Illustration 4

Table 4



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Illustration 5

Table 5



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Fig. 1:(on left) RF insert coil with animal heart and tuning console. The animal can beplaced in insert and kept alive by supply of oxygen and anesthesia. (On right) The vertical

bore magnet design of superconducting gantry operable at ultrahigh 900 mHz magnetic

field is shown. It is made of several layers of TXI CryoProbe walls directly in contact of

helium and liquid nitrogen. In center, hole is to place receiver-transmitter Rf insert coil to

place samples or animal [20].

Illustration 6

Figure 1



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Figure 2A: Ex vivo 3D MRI coronal T2 weighted MRI image TE/TR 45/2500 ms (on left

panel) with different skin structures is shown in a excised rat skin with drawn sketch to

show the sites of dermatopathy associated with gadolinium induced fibrosis [20].

Illustration 7

Figure 2a



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Figure 2B: Ex vivo 3D MRI coronal T2 weighted MRI image TE/TR 45/2500 ms (on left

panel) with different kidney structures is shown in a excised rat kideny with drawn sketch

to show the sites of fibrosis associated with gadolinium induced nephrogenic systemic

fibrosis (NSF). Kidney structures are shown in a rat kidney drawn to show the sites ofbasement membrane and nephrofibrosis. [26]

Illustration 8

Figure 2b



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Figure 3A: Different Gd contrast agents are shown to illustrate their noncovalent binding

sites with aryl structures of ligands and chelates by a phosphodiester link. Notice the size

of hydrophobic aryl structures and ligand chain length is determinant of tumbling effect

of hydrophilic Gd3+ moiety and increased relaxivity or better imaging contrast and MRI

signal intensity [26].

Illustration 9

Figure 3a



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Figure 3C: Sketch of Gd3+ complex having R1,R2,R3 ligands are shown in a cyclic arrangement to generate restriction

and torsion in the Gd3+ molecular complex adjacent to tissue collagen rich in hydrophilic amino acids. The interaction

of hydrophobic aryl ligands next to hydrophilic amino acids is basis of increased relaxivity and hydration moiety to

attract water or protons to create high proton MRI signal at the sites of accumulated Gd3+ concentration [26].

Illustration 10

Figure 3c



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Figure 4: Outline of gadolinium induced nephrogenic systemic fibrosis as sequence of

protein dysfunction

Illustration 11

Figure 4



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Figure 5A: Quantitative T2 maps were obtained from different concentrations of contrast

agents in seven solutions imaged in glass MR tubes. A sequence of increasing

concentrations of SPIOM is shown for the dephasing effect of SPIOM on decreased T2

MRI signal intensity (in left panel). A sequence of increasing concentrations of GdIII

DTPA is shown for the effect of gadolinium on increased MRI signal intensity(in middle

panel). The tubes demonstrate a marked T1 shortening effect by loading GdHPDO3A

(Omniscan) into apoferritin or GdIII-Apoferritin complex. After loading, the apofe

Physical Basis of Gadolinium Induced Skin Nephrofibrosis

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