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Physical Basis of Gadolinium Induced Skin Nephrofibrosis: Testing by Gadolinium-Protein Targeting Assay and Iron Oxide Nanoparticle Based Magnetic Resonance Microscopy

Rakesh Sharma1,2

1Center of Integrated Magnetic Resonance and Applied Research, National High Magnetic Field Laboratory, Tallahassee, FL 32304

2Center of Nanomagnetics and Biotechnology, Florida State University,Tallahassee, Florida 32304 USA

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 and kidney 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 image contrast, 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 fibrosis or fibrosing dermatopathy.

Key words: skin, kidney, MRI, 500 MHz NMR, 21 Tesla MR microscopy, spectroscopy, gadolinium, nephrogenic systemic fibrosis

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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 concern1 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. In case 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 mol/liter (gadodiamide; gadolinium-

diethylenetriamine-pentaacetatebismethylamide[Gd-DTPA-BMA], Magnevist® 0.5 mol/liter

(gadopentetate dimeglumine; Gd-DTPA), ProHance® 0.5 mol/liter (gadoteridol; Gd-HP-DO3A),

MultiHance® 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/liter (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

                                                            1 FDA. http://www.fda.gov/cder/drug/infopage/gcca/ 

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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].

Table 1: NSF Case Reports Associating the Gd-Chelate Administration with the incidence of NSF and biophysical factors[2-16] ____________________________________________________________________________________________ Reference Year No. of patients Contrast agent Tissue proteins MRI findings Gd-ligand used binding skin kidney ____________________________________________________________________________________________________________________________ Piera-Velzquez et al. 2010 12 GBCA SMA,Col1-A1 fibroblast Backer et al. 2010 23 GBCA ?? chronic kidney disease 5D Kribben et al. 2009 Martin et al. 2008 collagen basement membrane proteins Boyd et al 2006 1 N/A ↑magnetization, Gd in biopsy Marckmann et al 2006 13 Gadodiamide ?? ↑magnetization low dynamicity Grobner 2006 5 Gadodiamide ?? ↑magnetization ↑magnetization and relaxivity Broome et al 2006 12 Gadodiamide ?? ↑magnetization and relaxivity Sadowski et al 2007 13 Gadodiamide ?? Gd in biopsy Gadobenate dimeglumine 97% ↑magnetization Gadoxetic acid 50% ↑magnetization Deo et al 2007 3 Gadopentetate dimeglumine ?? ↑magnetization Khurana et al 2007 6 Gadodiamide ?? High et al 2007 7 N/A Gd in biopsy Lim et al 2007 2 Gadodiamide ?? Marckmann et al 2007 19 Gadodiamide ?? ↑magnetization and relxivity Gadodiamide ?? ↑magnetization and relxivity gadopentetate dimeglumine ?? ↑magnetization and relxivity gadobenate dimeglumine , 97% ↑magnetization and relxivity gadofosveset 91% Nikken et al. 2007 GBCA transglutaminase 2 (TG2), galactosidase, alkaline phosphatase, carboxypeptidase, carbonic anhydrase,

myeloperoxidase N/A = not available. SMA= smooth muscle action-1, Coll-A1 = Collagen A1

Gadolinium and Origin of Nephrogenic Fibrosing Dermopathy:

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 fibrosis due 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

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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 β), 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]. MMMMMMM

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 at low resolution. At high resolution 900 MHz MRI, each nephron

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 GBM barrier. 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

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to store large magnetic energy with permeability limit of 900 MHz magnet μ/μ0 =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 Nb3 Sn conductor coil 921

kg, weight of Nb3 Sn reinforcement coil 593 kg, weight of NbTi conductor coil 1273 kg, weight

of NbTi reinforcement coil 824 Kg at operating current of 290 ºA; inductance of coils 953 H;

stored energy 40 MJ; permeability limit for the 900 MHz magnet μ/μ 0 =1.020; 3. bucking coils

have Nb3 Sn-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.

Fig. 1:(on left) RF insert coil with animal heart and tuning console. The animal can be placed 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].

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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, protein substrate 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 (Δ1/T1 or Δ1/T2) s-1 per

millimolar concentration of MRI contrast complex normalized to the millimolar concentration of

the Gd contrast 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 (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

ligand 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 τR) 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 τR of Gd3+ is 50-100 picoseconds while τR of albumin is 50 ns.

The protons of Gd bound protein active groups precess at Larmor frequency proportional to

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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 effect Kd 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 profiles were first 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 coordination

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 ºA vs 3.1 ºA

makes more 50% difference of relaxivity ∆ri in coordinated water molecules. However, dipole

interaction with Gd3+ electron spins (Gd-proton distance in parallel or 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 + τm+ r1SS + r1OS (2)

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where q is inner sphere hydration number and τm 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 τm.

5. Protein binding with gadolinium determines the rotational 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 τm.

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 = Slopemax / ∆(1/T1)arterial (4)

where slopemax 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, Δ(1/T1) = r1[Gd]. One can increase the Gd payload as well as its

relaxivity.

GFR can be estimated using T1 measurements before and 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.

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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.

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].

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 of basement membrane and nephrofibrosis. [26]

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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].

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Figure 3B: The chemical basis of tissue protein binding with Gd –ligand structure is shown to play role in hydration state and tumbling of Gd contrast imaging complex structure in hydrophilic environment. Notice the restricted motion of Gd complex and increased relaxivity or enhanced magnetization to cause increased MRI signal. The nature of protein (collagen, ferritin, albumin) having specific amino acid aryl structure (shown as gray box) in chain influences the hydration state, excessive availability of free Gd3+ whose accumulation in tissue likely causes fibrosis due to disorientation of peptides of protein(s) in epidermis and renal dysfunction [26].

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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]. 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 accumulated sites 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:

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The nanoparticle SPIOM dephasing and MR signal relationship can be shown as: Signal = TE α 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 SPIOM induced myocardiac fiber specific field 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 with proteoglycans 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.

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

  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

explore new alternative with less risk of renal toxicity and fibrosis by measurement of skin and

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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 pre-

clinical study; 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 100–125 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 gift from 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’ ΔE.

The water proton T1 relaxation constants [1/T1obs]Gd-ferritin of 0.1 mM solution of Gd3+ with

different concentrations of ferritin (100-700 µg/mL) were measured at 20 MHz, 37°C keeping

relaxation constant [1/T1obs]Gd at same concentration of Gd.

Gd + Ferritin Gd-Ferritin

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

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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 µg/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 in

intravenous catheter (Abbocath Lab, IL) on nose with 30% 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.

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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 eosin staining [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 pre-

contrast 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 15) 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 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 [ Σa(p) = a(p)cortex+ medulla+pelvis] by following formula

VKid = 10 PS[Σa(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 stereological methods 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

Bowman’s 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

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tenth section 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, 1⁄2fa is the

fraction of the total section area used to count glomeruli, and Q is the actual number of glomeruli

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 hematoxylin-

eosin 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 spectroscopy

All 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.,

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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 pl1=3 dB; power level for

water suppression pl9=55 dB; power angle p1=7.5 lsec (90◦ pulse); power angle for water

suppression p12 = 60 ls; water suppression at O1 = 4.76 ppm; relaxation delay d1 = 12.85 sec

(5×T1); 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 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

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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 two-

sided 5% level of significance [26].

Results:

The titration plot in Figure 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 linearly with ferritin concentrations. Increased enhancement

factor E was 4.5 fold suggested the ferritin binding with Gd3+ considering 0.125 mM Gd3+ actual

concentration in solution. The measured r1 or 1/T1 includes three components as shown in

Equation 9.

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 apoferritin was extensively dialysed to remove free Omniscan from apoferritin solution, ensuring that the observed increase in relaxivity was due to GDIII-apoferritin complex. At different time intervals, Omniscan induced rat skin changes in epidermis and hair follicle structures are shown indicating possibility of Gd induced fibrosis at different time intervals of 0-8 hours (in right panel).

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Figure 5B: (Top row) Quantitative T1 map with pseudocolor; (middle row) A sequence of increasing concentrations of SPIOM is shown for the dephasing effect of SPIOM on decreased MRI signal intensity. (Bottom row) A characteristic NMR peak variation is shown at different concentrations of protein. Relaxation constants of different 200, 400 and 1000 μg/mL concentrations of SPIOM nanoparticles placed in three test tubes are shown as bars (A-C) at 25 ˚C; (D-F) at 35 ˚C and MR images (A-F). 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

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negative contrast of accumulated particles. The MRI signal intensity changed with concentration

of SPIOM as shown Figures 5A and 5B.

Gadolinium treated rats showed 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

enhanced images. 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.

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Figure 6: In vivo 500 MHz T2 weighted MRI image of whole animal is shown for normal, Gd enhanced image and SPIOM post-injected contrast enhanced image. Gd enhanced image showed bright regions of blood in arteries and kidneys shown in middle panel B. SPIOM enhanced image showed darker regions of iron oxide accumulated regions shown in panel C.

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 skin and 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 = (l×h×w) 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 not

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consistent. 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 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 µm, dermis 600 µm, hair follicle 50-70 µm, sebaceous gland

50-60 µm. Gadolinium treated skin epidermis measured 200 µm or more, dermis 750-800 µm rich in

collagen as shown in Figure VII (panel A). Hair body measured 500 µm long with 100 µm 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).

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Figure 7: Excised rat skin samples are shown with skin structures by histology from normal rat animal(panel A), gadolinium treated animal skin (panel B), and SPIOM treated animal skin (panel C). Note the thickened and disrupted epidermis by Gd treatment and SPIOM accumulation in epidermis and hair structures with dephasing effect appeared dark.

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 Tesla MRI images.

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Figure 8: (in first row) 21.1Tesla MRI microimaging of excised rat kidney is shown K (appendix gland A on top of kidney). In middle panel, different structures of renal cortex, medulla, calyx, pelvis, and pyramid are shown by arrows after gadolinium treatment. On right, a spin echo image of rat kidney with brightness after Gd treatment and dark medulla region after SPIOM is shown. Different structures show………1, 2, 3, 4. (in second row) Ex vivo MR images by GE technique using cationic GdIII-ferritin as a contrast agent. The graphs show the signal intensity profiles of a line through tissue (on left) in normal (■) and SPIOM injected (Δ) tissues (n=7) and averaged spatial power spectrum of intensity profiles with bars (mean +1 sd) on right.

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 µm distance in two points on x axis as dark spots shown in Figure VIII. The accumulation of SPIOM in kidney cortex was detected with MRI in vivo after 5 intravenous injections. Kidney slices in 100 x 500 µm 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 µm) 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.

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Ex vivo 900 MHz MRM isotropic images showed <15 microns plane resolution and isotropic 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 kidney protein 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 µm (at 500 MHz) and better at ex vivo 900 MHz 15 µm isotropic kidney images. Comparison of post-Gd MRI images with histology digital images showed two distinct 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 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).

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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 μm;

medulla 400 μm; 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 rich regions 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).

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Figure 9: Effect of gadolinium on right and left kidneys are shown with different renal structures by histology (First row); proton density and T2 weighted 21 Tesla MRI images of normal kidneys(Second row); proton density and T2 weighted 21 Tesla MRI images of gadolinium treated kidneys (third row). Proton density and T2 weighted 21 Tesla MRI images of Gdolinium treated vs SPIOM treated kidneys are shown in fourth row to illustrate the difference in MRI signal intensities by gadolinium enhanced relaxivity and SPIOM induced dephasing effect. At bottom, a high power histology section shows cytomorphic details of different structures in kidney. Cortex-medulla thickness is marker of nephrogenic fibrosis (shown as M-C). Nephron fibrosis was distinct and contrast agent Gadolinium and SPIOM contrast agent accumulate in kidneys as shown in panels at bottom.

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Table 2: Comparison between histology and MRI measurements of dermal and renal structure size in normal, gadolinium treated and SPIOM treated excised tissues shown in Figures 3 and 6.

______________________________________________________________________________

Measurement Histology MRI (in mm) ____________________________________________ Normal Gd-enhanced SPIOM (A) (B) (C) (D) ______________________________________________________________________________ SET 0.20 + 0.04 0.23 + 0.08 0.36 + 0.08 0.26 + 0.05 Hair follicle 0.48 + 0.06 0.58 + 0.05 0.80 + 0.08 0.65 + 0.08 Cortex 1.23 + 0.30 1.85 + 0.45 2.65 + 0.62 2.15 + 0.35 Medulla 2.25 + 0.55 2.95 + 0.68 3.55 + 0.35 3.35 + 0.40 Pyramid size 13.55+2.50 16.50+3.80 18.55+5.50 15.55+5.50 ______________________________________________________________________________ (A) vs (B) P value < 0.001; (A) vs (C) P value <0.001; (A) vs (D) P value <0.05 (B) vs (C) P value <0.05; (B) vs (D) P value <0.05; (C) vs (D) P value <0.05.

Table 3: A comparison of proton density and T2-weighted MRI visible structures of excised skin and excised kidney is shown in normal, post-Gd and SPOIM treated specimens as quick

guide by gross evaluation.

MRM features PD T2 weighted GdIII enhancement SPIOM

Hair follicle Bright Brighter; ++ Dark Brighter; +++ Epidermis Brighter;++ Brighter; +++ Brighter; ++ Dark Dermis Bright;+ Gray Brighter; + Dark Sebaceous gland Bright Hypointense; -- Brighter; ++ Dark Stratum corneum Isotense Dark gray Isointense Brighter; ++ Cortex Gray Bright White Band Bright Medulla Isotense Dark stripe Isotense Dark Pelvis Gray Hyperintense Bright Gray

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

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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.

Table 4: Measurement of Skin and kidney structures by MRI and Histology in gadolinium treated and SPIOM treated rats in evaluation of fibrosis.

   Change in structure (in % over nomal size)  

                         Gd                              SPIOM 

        histology of dermatopathy and fibrosis 

              Skin                                              Kidney 

Epidermis 2.5 -5 fold none Dermis +10-15 none Hair follicle +30 none Oil gland +20 invisible Cortex -10 none White band +50 invisible Medulla -20 +20 Pelvis +20 invisible

clefts, ulceration in epidermis wide pelvis, subcapsular band wide widening of hair follicle (x2) hypointense medulla (-40%) brighter oil glands (+30%) accumulated contrast agent (-80%) dermal fibrosis swelling of pyramids(+1.5-2.0 fold) acanthosis

 

Table 5. Absolute concentrations of major endogenous metabolites quantified from normal, Gd-treated and SPIOM treated kidney extracts by 1H-NMR. Metabolites are shown as chemical shift in ppm.

Metabolite Assignment Concentration(µmol/g) (ppm) ____________________________________________________________ Normal Gd-treated SPIOM treated Monounsaturated fatty acids (MUFA)/glycogen 5.42 5.35 ± 0.75 13.40 ± 2.10 5.24 ± 1.85 Allantoin 5.38 2.35 ± 0.15 12.35± 2.60 10.5 ± 2.50 Glucose 5.25 + 4.64 12.87 ± 0.40 6.50 ± 0.60 8.50 ±1.50 Triacylglycerol (TAG) 4.16 10.17 ± 4.17 12.50 ± 3.00 10.15± 2.00 Inositol 4.06 2.75 ± 1.10 2.65 ± 1.35 2.50 ± 1.50 Glycerol-6-phosphate 3.99 3.00 ± 0.15 2.80 ± 0.30 2.50 ± 1.50 Phosphatidylcholine 3.67 3.13 ± 0.42 7.55 ± 1.55 3.20 ± 2.52 Taurine 3.43 4.90 ± 1.20 6.80 ± 2.00 4.55 ± 3.20 Trimethylamine-N-oxide(TMAO)/CH3 3.27 1.16 ± 0.21 (+betaine) 13.5 ± 2.85 1.20 ± 1.55 Betaine/CH3 3.25 1.15 ± 0.20 (TMAO) 8.55 ± 1.00 1.20 ± 0.30 Glycerophosphocholine 3.23 1.00 ± 0.11 9.45 ± 2.45 0.78 ± 0.30 Cholines 3.20 14.23 ± 2.00 10.4 ± 2.55 6.50 ± 1.00 Phosphatidylethanolamine 3.10 1.15 ± 0.22 5.55 ± 1.32 1.00 ± 0.50 Creatinine+creatine 3.02 − 3.04 0.79 ± 0.20 0.39 ± 0.15 0.62 ± 0.35 Asparagine 2.96, 4.00, n.d 0.65 ± 0.15 n.d. Trimethylamide 2.88 10.0 ± 0.02 7.85 ± 1.32 8.55 ± 1.20 Polyunsaturated fatty acids (PUFA) 2.85 78.50 ± 13.00 48.55 ± 5.5 115.20±12.45 Citrate/Aspartate 2.80 + 2.65 0.91 ± 1.15 1.55 ± 3.45 2.50 ± 2.25 Dimethylamide 2.72 0.06 ± 0.05 n.d. n.d. Glutathione (GSH) 2.63 1.85 ± 0.20 2.54 ± 5.40 7.55 ± 2.40 Glutamine 2.46 1.05 ± 0.20 1.00 ± 0.05 1.10 ±0.05 Pyruvate 2.38 1.40 ± 0.60 1.50 ± 0.10 2.00 ± 0.50 Glutamate 2.36 3.00 ± 0.57 2.55 ± 0.35 2.50 ± 0.55 Acetate 1.96 0.95 ± 0.10 1.00 ± 0.55 0.80 ± 0.05 Lysine/Arginine 1.7 25.5 ± 0.50 15.5 ± 0.55 10.0 ± 0.50 Citrulline 1.58, 3.15 1.80 ± 0.55 3.00 ± 1.00 2.65 ±0.55 Alanine 1.45 2.50 ± 0.20 1.85 ± 0.30 1.80± 1.00 Lactate 1.33 3.05 ± 0.30 11.00 ± 2.25 5.50 ± 2.00 3-OH-butyrate 1.15 n.d. 1.5 ± 0.05 3.50 ± 1.00 Valine+leucine+isoleucine 0.99 − 1.05 3.15 ± 1.50 3.00 ± 0.55 2.50 ± 0.50 Total triglycerides (TG) 0.91 0.90 ± 0.20 0.50 ± 0.50 0.45 ± 0.30 Cholesterol 0.5-0.8 6.90 ± 1.00 15.5 ± 5.5 16.50 ± 1.00 n.d., nondetectable. TMAO and betaine concentrations are based on three CH3 groups.

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Figure 10: Representative 1H-NMR of kidney extracts. (A) Normal kidney (NAT). (B) Post Gadolinium Eight-hours treated kidney (Gd-8). Peak identification was performed using two-dimensional 2D-H,C-HSQC NMR, which visualized different NMR resonance peaks for the exact structure analysis (chemical shifts for glucose: r1H = 5.25 ppm; allantoin: r1H = 5.39 ppm; and glycogen: r1H = 5.45 ppm).

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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 artery 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 = EF*RBF*(1 – Hct), where RBF is renal

blood flow measured by phase contrast flow quantification and Hct is measured by

hematocrit.[35]; 3. Using contrast enhanced 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.

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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 generate sufficient MRI visible signal intensity. Perhaps, extra 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 as filtration 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 α1/2 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]. The effect

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

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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 Omniscan® gadolinium skin exposure. Earlier study showed

accumulation of gadolinium in skin 1.7 + 0.2 µmol/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 slice thickness 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 the

cost of active energy in active filtration against pressure gradient. Omniscan with excessive

gadolinium and caldiamide ligand and low thermodynamic stability was reported to have NSF-

like 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

Rakesh Sharma                   

35 

 

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:

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 α1/2 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 β, Transglutaminase in skin targeted by

gadolinium imaging contrast agents. Cationic superparamagnetic iron oxide (SPIOM; Fe3O4

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 and biodegradable 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

Rakesh Sharma                   

36 

 

basement membrane [1]. The accumulated nanoparticle sites visualized (dark hypointense

region) the basement membrane breakdown down to the below 15 micrometers scale using 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

Rakesh Sharma                   

37 

 

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 physiologic information.

Slope of gadolinium DTPA enhancement 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

The type of ligand in gadolinium chelates bound with tissue proteins may play role to cause

gadolinium toxicity resembling with NSF. MRI visualizes skin and renal tissues with oil or fat

glands or intracellular fat. MRI is highly sensitive to skin epidermis and hair follicle to measure

Rakesh Sharma                   

38 

 

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.

Acknowledgements

Author acknowledges the supply of nanoparticles from Y. Haik at Center of Nanomagnetics and

Biotechnology and chemical-biomedical engineering 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 and suggestions. 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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