Nanosized Contrast Agents to Noninvasively DetectKidney Inflammation by Magnetic Resonance ImagingJoshua M. Thurman and Natalie J. Serkova

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Several molecular imaging methods have been developed that use nanosized contrast agents to detect markers of inflamma-

tion within tissues. Kidney inflammation contributes to disease progression in a wide range of autoimmune and inflammatory

diseases, and a biopsy is currently the only method of definitively diagnosing active kidney inflammation. However, the devel-

opment of new molecular imaging methods that use contrast agents capable of detecting particular immune cells or protein

biomarkers will allow clinicians to evaluate inflammation throughout the kidneys and to assess a patient’s response to immu-

nomodulatory drugs. These imaging tools will improve our ability to validate new therapies and to optimize the treatment of

individual patients with existing therapies. This review describes the clinical need for new methods of monitoring kidney in-

flammation and recent advances in the development of nanosized contrast agents for the detection of inflammatory markers

of kidney disease.

Q 2013 by the National Kidney Foundation, Inc. All rights reserved.Key Words: Nanoparticle, Kidney, Imaging, Inflammation, Magnetic resonance imaging

Introduction

Inflammation is central to the pathogenesis of a widerange of acute and chronic kidney diseases. The accurateassessment of inflammatory processes within the kidneysimproves our understanding of kidney disease pathogen-esis, and it improves our ability to treat individual pa-tients. For example, the treatment of most forms ofglomerulonephritis involves immunosuppressive drugs,and there is evidence that other kidney diseases mayalso respond to immunomodulatory drugs. However,all immunosuppressive drugs increase the risk of infec-tion and have to be used with caution. Therefore, the de-tection of ongoing kidney inflammation can guide the useof these medications.

Many new drugs for modulating or blocking the im-mune response have been developed in recent years, andthese new agents have led to significant improvements inoutcomes for some kidney diseases. For example, Rituxi-mab is effective for the treatment of several types of kidneydisease.1-4 Someof the newer agents have a narrower rangeof action and may be less immunosuppressive than olderdrugs such as cyclophosphamide, but patient selection isvery important given the more focused biologic actions ofthese drugs. Although several tests of the blood and urinecan be helpful in diagnosing the underlying disease,nephrologists are still heavily dependent on kidneybiopsies to determine the etiology and activity of the

m Department of Medicine, University of Colorado–Denver School of

ne, Aurora, CO; and Department of Anesthesiology, University of Col-

Denver School of Medicine, Aurora, CO..T. is a paid consultant for Alexion Pharmaceuticals, Inc.

dress correspondence to Joshua M. Thurman, MD, Division of Renal

es and Hypertension, Department of Medicine, University of

do–Denver School of Medicine, Campus Box B115, Aurora,045. E-mail: Joshua.thurman@ucdenver.edu

013 by the National Kidney Foundation, Inc. All rights reserved.

8-5595/$36.00p://dx.doi.org/10.1053/j.ackd.2013.06.001

Advances in Chronic Kidney Disease, Vol 2

underlying disease. Conventional radiology does notusually weigh heavily in treatment decisions.

In recent years there have been significant advances infunctional and molecular imaging methods. In additionto anatomic evaluation of the kidneys, these new tech-niques can provide quantitative evaluation of kidneyfunction (eg, kidney blood flow and glomerular filtrationrate [GFR]). ‘‘Molecular imaging’’ methods can be used tononinvasively detect specific molecules of interest withintissues, and nanoparticles are a useful platform for devel-oping molecular imaging contrast agents. They are smallenough to penetrate most tissues, they can be designedfor detection by standard radiologic methods, and theycan be linked to targeting proteins that direct the nano-particles to specific molecular markers. Superparamag-netic iron oxide (SPIO)-based nanoparticles have beenused as magnetic resonance imaging (MRI) contrastagents to detect macrophages in animal models of kidneyischemia and in kidney transplant recipients. More re-cently, targeted SPIO nanoparticles have been used asmolecular imaging contrast agents to detect complementactivation in preclinical models of glomerulonephritis. Inthis review, we will discuss the use of SPIO-based con-trast agents and T2-weighted MRI to detect and monitorkidney inflammation.

The Clinical Need for Imaging Biomarkers ofKidney Inflammation

Clinicians are typically alerted to the presence of kidneydisease by the detection of elevations in the serum creat-inine or inappropriate substances in the urine (eg, pro-teinuria or red blood cells). Patients may developphysical exam findings, such as peripheral edema orsigns of uremia, but these are often late-stage manifesta-tions of disease and are nonspecific. Once a disease isbroadly categorized (eg, acute kidney injury [AKI], ne-phrotic syndrome, glomerulonephritis), specific bloodand urine tests may help to find the etiology of disease.

0, No 6 (November), 2013: pp 488-499

Nanosized Contrast Agents to Detect Kidney Inflammation by MRI 489

However, for most types of kidney disease, the availablebiomarkers are not sufficient to make an early or defini-tive diagnosis without performing an invasive biopsyprocedure. Improved biomarkers are desperately neededfor several different kidney diseases and clinical syn-dromes, and detection of inflammatory markers withnanosized contrast agents may transform the care of var-ious kidney diseases in the near future.

Lupus Nephritis

Lupus nephritis is the prototypical immune-complex glo-merulonephritis. Kidney injury is caused by the deposi-tion of immune complexes in the glomerulus withactivation of the complement systemon kidney structures.There is uncertainty as to whether the immune system isresponding to kidney antigens or whether the kidney sim-ply represents the site of immune-complex deposition. Itshould also be noted that immune complexes are notprominent in some histologic patterns of lupus nephritis.5

Nevertheless, all forms of lupus nephritis are broadly cat-egorized as ‘‘autoimmune’’ and are treated with immuno-

CLINICAL SUMMARY

� Currently, a renal biopsy is necessary for the definitive

diagnosis of renal inflammation.

� Molecular imaging contrast agents enable the noninvasive

detection of tissue biomarkers of inflammation.

� Nano-sized contrast agents can be targeted to

inflammatory markers.

� Nano-sized contrast agents can serve as imaging

biomarkers of renal inflammation.

suppressive drugs.Once a definitive diagno-

sis is made (generally by bi-opsy), patients are started oncourses of therapy that maylast several years.6,7 Lupus isa notoriously heterogeneousdisease, and fewer than 50%of patients treated with thestandard therapies enterremission within the first6 months of therapy.7-10

Furthermore, a biopsysamples only a small portion

of the kidney. Diseases such as lupus are often focal, andpatients can be staged incorrectly because of samplingerror of the biopsy. For example, it has been estimated bymathematical modeling that in a biopsy that contains 20glomeruli, 14 of the glomeruli need to show diseaseinvolvement to conclude that there is involvement ofmore than 50% of the glomeruli (diffuse disease).11 Obvi-ously, the fewer glomeruli sampled in the biopsy, thegreater the risk of misclassifying the disease.

Because of the variable response to treatment in pa-tients with lupus nephritis, clinicians must repeatedlyre-evaluate a patient’s clinical condition. During a pro-longed course of therapy, clinical and laboratory findingsare used to determine whether a patient is responding totherapy and the treatment should be continued. For pa-tients who do not respond to treatment, the decisionmust be made as to whether the treatment intensityshould be increased or, conversely, whether damage tothe kidney is irreversible and treatment should be discon-tinued. Common biomarkers of disease activity in lupus

nephritis include the degree of proteinuria, the number ofred blood cells seen in a spun urine sample, serum anti-double-stranded DNA antibodies, and the level of C3 inplasma. All of these biomarkers have limited accuracyfor determining the degree of kidney disease activity orthe degree of irreversible kidney damage.12 In one report,information obtained from a repeat biopsy at the end ofinduction treatment was predictive of a doubling of se-rum creatinine whereas no clinical or laboratory parame-ters were predictive of this outcome.13 Thus, the biopsy isthe currently the best method of judging the severity ofa patient’s disease and their response to therapy, andthe persistence of immune deposits in a second biopsyis one of the strongest predictors of disease progression.Therefore, the ability to noninvasively detect these de-posits in tissues could provide a powerful method for tai-loring a patient’s treatment.

Other Forms of Immune-Complex Glomerulopathy

Other glomerular diseases associated with immune-complex deposits are frequently treated with immuno-

suppressive drugs. Forexample, type 1 membrano-proliferative glomerulone-phritis, IgA nephropathy,and membranous diseaseare characterized by glomer-ular deposits of immuno-globulin and complementproteins. The M-type phos-pholipase A2 receptor wasidentified as the target anti-gen for most patients withidiopathicmembranous dis-ease,14 raising the pos-

sibility that antibodies to this protein can be used asa biomarker of the underlying immune process. The ti-ter of antibody specific to this receptor may be usefulfor monitoring the response of patients to treatmentwith immunomodulatory drugs,15 although tests forthis antibody are still not widely available. Urinary pro-teomics has revealed disease biomarkers for otherforms of glomerulonephritis, but these tests are of lim-ited use and have not entered clinical practice.16,17

Therefore, for most forms of glomerulonephritis, goodnoninvasive biomarkers of disease activity have notyet been developed.

Other Chronic Inflammatory Diseases of theKidney

Not all chronic inflammatory diseases of the kidney arecaused by immune complexes. For example, C3 glomerul-opathy is a recently described pattern of kidney injury de-fined by the detection of glomerular C3 in the absence of

Thurman and Serkova490

glomerular immunoglobulin deposition.18,19 The effectsof corticosteroids and standard immunosuppressivedrugs on this disease are not clear.19 Eculizumab, a thera-peutic complement inhibitor, may be effective in some pa-tients.20 C3 glomerulopathy is clinically a veryheterogeneous disease. Methods to noninvasively detectimmune deposits in the kidney would greatly facilitatethe evaluation of new and existing treatments withoutthe need for serial biopsies. Given that this disease is de-fined by the detection of glomerular immune deposits,the detection of these factors by molecular imaging couldsomeday replace the biopsy for disease diagnosis.

AKI

AKI can be caused by awide range of hemodynamic, toxic,infectious, andmetabolic insults to the kidneys.21 In recentyears there has been an intensive effort to discovernew, early biomarkers of AKI.22-24 AKI is increasinglyunderstood to be an inflammatory disease.25,26 Someinflammatory cytokines are detected early in the courseofAKI,27 although thesemarkers are not disease or tissuespecific. Molecular imaging methods have been devel-oped to detect tissue inflammation inmodels of AKI (dis-cussed in Functional and Anatomical Kidney Imaging).Unfortunately, these methods require 24 to 48 hours,and a key goal of detecting inflammation in patients atrisk of AKI is to stratify patients to early interventions.Thus, the role of molecular imaging in the diagnosisand staging of AKI will require the development ofmore rapid imaging methods.

Transplant Rejection

Kidney allograft rejection can occur at any time aftera kidney transplant and is usually detected by an increasein serum creatinine. Treatment of rejection generally in-volves escalation of a patient’s immunosuppressive treat-ment. Therefore, rejection must be distinguished fromnonimmunologic causes of injury, such as BK virus ne-phropathy.28 Several assays show promise as biomarkersfor distinguishing rejection from other causes of allograftfailure, although none are yet in clinical use.29 Conse-quently, transplant biopsies are currently necessary foraccurately detecting immunologic rejection as a causeof allograft dysfunction.

Functional and Anatomical Kidney Imaging

Abnormal kidney function is the most common indica-tion for kidney imaging. Advances in radiological sci-ences and nuclear medicine have led to an enhancedrepertoire of imaging modalities and endpoints that canbe applied and observed, respectively, to delineate theunderlying abnormality. Kidney imaging encompassesfourmain techniques: ultrasound (US), computed tomog-raphy, MRI, and nuclear medicine (including positron

emission tomography and single-photon emission to-mography [SPECT]).30-33 Modern anatomical techniquesallow for a superb soft-tissue contrast (MRI) and spatialresolution (MRI and computed tomography) whereasfunctional (also called ‘‘dynamic’’) scans allow for preciseassessment of excretion rates, glomerular filtration,tubular concentration and transit, blood volume, perfu-sion, and oxygenation (Doppler US, gadolinium [Gd]-enhanced MRI, blood oxygen level-dependent [BOLD]MRI, 99mTc-MAG3 SPECT, 123I- or 131I-hippuran SPECT).Nevertheless, although various clinical indications can beinvestigated by a particular imaging protocol (Table 1),there is no existing, validated imaging platform to detectkidney inflammation.

Anatomical and Functional MRI

Current MRI protocols are able to display morphologicalinformation on kidney parenchyma and vessels as well asfunctional data, such as perfusion, filtration, diffusion,and oxygenation. MRI has the best soft-tissue contrastamong all imaging techniques, even without the use ofintravenous contrast. Because MRI uses complex physicsto generate images (pulse sequences), various parameterscan be used to optimize assessment of specific anatomic,morphologic, and functional endpoints:

- Renal cell carcinoma, angiomyolipoma, and kidneycysts (Fig 1A) can be readily distinguished by anatomi-cal T1- or T2-weighted MRI.31,34

- Although the measurement of GFR by MRI is challeng-ing, Gd-enhanced T1-weighted MRI protocols havebeen developed for the relative assessment of kidneyfunction (Fig 1B). These dynamic contrast-enhancedMRI protocols require sampling of the abdominal aortaand both kidneys with a sufficient time resolution(,2 seconds) to accurately define the arterial inputfunction and to separate the cortical vascular phase(perfusion) and the filtration rate of the Gd con-trast.30,33,35

- The BOLD MRI technique does not measure pO2 di-rectly but allows for intrarenal R2* (relaxation rate)measurements, which are closely related to the concen-tration of deoxyhemoglobin.32,36 Static comparison ofR2* values in both kidneys by BOLD MRI can identifyhypoxia in one kidney (eg, due to renal arterystenosis).37,38

- In acute kidney injury, direct ischemic damage to thecells may lead to apoptosis or necrosis of tubular cells.Diffusion-weighted MRI is useful to separate cellularedema (reversible damagewith decreased apparent dif-fusion coefficients) from cellular kidney necrosis (irre-versible damages with increased apparent diffusioncoefficients).36

- Kidney fibrosis involves excess extracellular matrixsynthesis accompanied by increased abundance of fi-brillar collagens. Fibrosis is generally considered an

Table 1. Existing Imaging Modalities and Their Advantages and Disadvantages for Kidney Imaging

Imaging Modality

Spatial

Resolution Clinical Problem Advantages Disadvantages

US 5-10 mm Diffuse kidney diseases

Kidney mass lesions

Kidney cysts

Urinary tract obstruction

Kidney stones

Hematuria

Transplanted kidney

Doppler US:

Vessel patency

Abnormal vascularity

Renal artery stenosis

Real-time nature of US highly suited for

kidney biopsy and interventional

procedures

High potential for functional Doppler

imaging

Microbubbles as new contrast agent

Inexpensive

Low potential for molecular imaging

Suboptimal image quality in obese patients

CT 5 mm Kidney trauma

Kidney cysts

Kidney carcinoma

Ureteric calculi

High spatial resolution

Fast (in a single breath-hold) acquisitions of

the whole abdomen

Low potential for molecular imaging

Low potential for functional imaging

Contrast is required (toxicity)

Radiation exposure

MRI 5 mm Kidney cysts

Kidney carcinomas

Kidney functions

Kidney transplantation (living kidney donors)

MRA:

Renal artery stenosis

Abnormal vascularity

High spatial resolution

Superb soft-tissue contrast

No ionizing radiation

High potential for functional imaging

Moderate to high potential for molecular

imaging

Moderate to high costs

Prolonged scans

Complex physics

Pacemakers and metal clips are

contraindicated

Nuclear medicine

(PET and SPECT)

10-15 mm Kidney failure

Kidney obstruction99mTc-DMSA: kidney scarring99mTc-DTPA and MAG3: GFR assessments

Supreme functional (rather than anatomic)

imaging;

High potential for molecular imaging

Low spatial resolution

High costs

Radiation

Abbreviations: CT, computed tomography; GFR, glomerular filtration rate; MRA, magnetic resonance angiography; MRI, magnetic resonance imaging; PET, positron emissiontomography; SPECT, single-photon emission tomography; 99mTc-DMSA, dimercaptosuccinic acid; 99mTc-DTPA, diethylene-triamine-pentaacetate; US, ultrasound.Summarized based on previously published radiologic-based reviews.32,33,76

Nanosized

Contrast

Agents

toDetect

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Inflam

mation

byMRI

491

Figure 1. DCE-MRI of bladder and kidney (images are presented at 0, 2, 6, and 14min of Gd-bolus injection) of control and 5/6nephrectomymice. Decreased enhancement in the bladder and increased enhancement in the kidney are present in nephrec-tomy animals, suggesting decreased filtration and excretion of Gd after surgery (N.J.S. unpublished data). Abbreviations:DCE-MRI, dynamic-contrast enhanced MRI; Gd, gadolinium; MRI, magnetic resonance imaging.

Thurman and Serkova492

irreversible process that is unresponsive to treatmentwith immunosuppression and portends progression toESRD. Recent studies have reported on the diagnosticpotential of magnetic resonance (MR) elastographyand diffusion-weighted and diffusion-tensor MRI asnoninvasive methods to detect kidney fibrosis inESRD and transplanted kidneys.39,40

MRI Contrast

Diagnostic MRI routinely uses contrast agents to alter therelaxation rate of water protons because the signal inten-sity in MRI is dependent on the concentration of water inthe area of interest. Effective contrast agents must havea strong local effect on either the T1- or T2-relaxationtimes, thereby shortening the relaxation time of the waterprotons. Two commonly used classes of MR contrastagents include paramagnetic T1-shortening contrastagents (Gd, manganese) and superparamagnetic T2-shortening contrast agents (iron oxide). The water mole-cules bound to these high-spin metals relax orders ofmagnitude faster than free water, resulting in the desiredchanges in signal intensity. However, Gd and iron oxidediffer in their MRI effects: Paramagnetic Gd has predom-inant T1-effects producing a positive contrast/brightimage on T1-weighted MRI due to shortening of the T1-values whereas superparamagnetic iron has a prevalentT2-effect and produces a negative contrast/darker sig-nals on T2-weighted MRI due to a reduction in T2-values (Fig 2A).

The most commonly used intravenous MRI contrastagents are Gd-chelates. All U.S. Food and Drug Ad-ministration (FDA)-approved Gd-chelates are low-molecular-weight contrast agents. Because they arefreely filtered by the glomeruli at first pass withoutany tubular secretion or reabsorption, they can be

used as glomerular filtration markers (see previous dy-namic contrast-enhanced MRI application descrip-tion).41 Free Gd is unfortunately toxic, and the stabilityof chelated Gd is inadequate in patients with ESRD be-cause of prolonged circulation times. In such cases,nephrogenic systemic fibrosis has been reported in asso-ciation with Gd use for MRI, and all Gd-chelates are con-traindicated in patients with ESRD, AKI, and Stage 4 to 5CKD.42,43

Iron oxide (SPIO nanoparticles) decreases spin-spinT2-relaxation times, resulting in negative contrast (tis-sue darkening) on T2-MRI. Nanoparticle imaging agentsare small enough to stay in colloidal solution and to pen-etrate tissues; however, they maintain physical char-acteristics that make them detectable by standardradiologic methods. In addition, nanoparticles can beeasily functionalized (a targeted moiety can be easilyadded to the iron-oxide-containing core). Most impor-tantly, unlike Gd, iron is a naturally occurring elementin human bodies and is taken up and metabolized bythe reticuloendothelial system, Kupfer cells, and macro-phages.44 Because of their natural metabolic fate, SPIOnanoparticles have been clinically used as liver contrastagents (2 FDA-approved agents, Feridex and Resovist)and as an intravenous iron supplement in anemia pa-tients (various FDA-approved agents, including Feru-moxytol, which is often used off-label for MRI)(Table 2).45 A very attractive feature of MRI is its quan-titative nature. The quantitative endpoints for T2-based MRI sequences include (but are not limited to)the precise calculations of apparent diffusion coeffi-cients (as mm2/second) in diffusion-weighted MRI (byvarying b-values), and—significant for nanoparticle ap-plications—T2-relaxation times (in milliseconds) byvarying echo times (TE) in T2-based sequences. The fol-lowing equation is applied for precise calculations of

Figure 2. Contrast enhancement of the kidneys using nanosized contrast agents. (A) Effects of T1- and T2-contrast agents onMRI signal intensity. (B) SPIO-labeled mesenchymal stem cell homing in rat kidney. Adapted from Ref.54 (C) Time course ofFerumoxytol accumulation in muscle, kidney, and liver as detected by decreased T2-times at various time points after injec-tion with the agent (N.J.S. unpublished data). Abbreviations: MRI, magnetic resonance imaging; SPIO, superparamagneticiron oxide.

Nanosized Contrast Agents to Detect Kidney Inflammation by MRI 493

T2-relaxation time as a function of signal intensity andTE values of each T2-MR image:

S ¼ M0

�12e2TR=T1

�e2TE=T2 (1)

S ¼ C2e2TE=T2 (2)

where C2 ¼ M0(1 – e-TR/T1) is a constant that gets fitted.Darkening of inflamed tissues (which correlates with

macrophage accumulation) after injection of these com-mercially available SPIO nanoparticles has been observedin animal models of focal ischemia; neuroinflammation;atherosclerotic plaques; heart transplants; kidney inflam-

mation; and, recently, cancer.46-52 The degree ofdarkening can be quantitatively assessed by measuringthe T2-value within a region of interest (eg, the renal cor-tex) before and after injection of the contrast agent.46,47,53

The change in T2-value reflects the abundance of SPIOthat has accumulated and thus reflects the abundanceof the cells or target to which the SPIO is bound. The abil-ity of nanoparticles to target different locations withinthe glomerulus depends upon their size and physico-chemical properties. An accompanying paper in this is-sue by Zuckerman and Davis53a describes nanoparticleswith different compositions, and their ability to targetthe glomerulus.

Table 2. Commercial SPIO Nanoparticle Formulations and Their Properties

Agent Trade Name Application Particle Size

Ferumoxsil Lumirem, Gastromark Oral SPIO for MRI .300 nm

Ferumoxide Feridex IV SPIO for MRI 80-150 nm

Ferucarbotran Resovist IV SPIO for MRI 62 nm

Ferumoxtran Sinerem, Combidex IV USPIO for MRI 20-40 nm

Ferumoxytol Feraheme IV USPIO for anemia treatment 18-30 nm

Abbreviations: IV, intravenous; SPIO, superparamagnetic iron oxide; USPIO ultra-small paramagnetic iron oxide.Summarized from Wang YX, Quant Imaging Med Surg. 2011;1:35-40.45

Thurman and Serkova494

Preclinical Studies Using Nanoparticles to DetectKidney Inflammation

Given the great clinical need for methods of noninva-sively detecting kidney inflammation, several studieshave used nanoparticles to detect specific immune cellsor immune proteins within the kidney. These studieshave been successful at detecting inflammatory markerswith good sensitivity. Furthermore, they have been usedto localize the sites of inflammation within the kidney.

Studies Using SPIO-Labeled Cells

Studies have used in vitro iron-labeling of various pro-genitor cells with subsequent grafting and in vivo MRIvisualization of labeled cells in the animal. Labeled mes-enchymal stem cells (MSCs) were observed in vivo in therat kidney cortex as long as 7 days after injection into therenal artery of healthy rats in a 1.5-T MR field54 (Fig 2B).Another study reported the glomerular homing of iron-stained MSCs in a rat model of mesangiolysis.55 Afterintravenous injection of SPIO-MSCs, reduced T2-signalintensity was observed in the cortex of pathologic kid-neys 6 days after injection. In this study, no loss of T2-signal was seen in the kidneys of control animals. Othergroups have reported similar findings using labeledMSCs in rat models of acute ischemia and AKI causedby glycerol injection.56-58 The persistent loss of T2/T2*-weighted signal was observed up to 14 days after injec-tion of SPIO-labeled MSCs (range 72 hours to 14 days).More recent studies have reported kidney localizationof SPIO-labeled macrophages in rat kidney transplantand mouse ischemia/reperfusion models.59,60 Animal4.7-T MR scanners were used in both studies. Negativecontrast of the kidneys was observed 24 hours afterSPIO-macrophage administration in the rat recipients ofallogenic transplants (5-days posttransplant), and thelow T2*-signal intensity zones corresponded to the distri-bution of SPIO-labeled macrophages by histopathology.No changes in T2*-weighted MRI were seen in the synge-neic allograft group. Another study labeled macrophageswith 150-nm SPIOs ex vivo.60 The left kidney of Balb/cmice was clamped for 45 minutes, and after 24 hours ofreperfusion the mice were injected with 2 3 106

nanoparticle-labeled macrophages or with the nanopar-ticles. SPIOs of this size are primarily taken up by the

reticuloendothelial system (not tissue macrophages);therefore, direct injection with the nanoparticles was per-formed as a control. In the postischemic kidneys, the in-jection of the labeled macrophages caused a discretedarkening between the outer and inner stripe of the outermedulla by T2-weighted images. A negative contrast ef-fect was not seen in the control kidney or in ischemic kid-neys injected with the control SPIOs.

Studies Using Untargeted Nanoparticles

Studies have also shown that immunocompetent cells(tissue-associated macrophages) can be detected by MRIin vivo after SPIO injection without preexisting ex vivocell labeling. Macrophages, virtually absent in normalkidney, may infiltrate kidney tissues in specific nephrop-athies such as various forms of glomerulonephritis, kid-ney allograft dysfunction (rejection or acute tubularnecrosis), and in acute ischemia/reperfusion injury. Asmentioned above, iron oxide nanoparticles are avidlycaptured by macrophages and induce a significant de-crease in the T2/T2* of affected kidneys. Plasma clear-ance and the route of excretion depend on the particlesize. The ultra-small superparamagnetic iron oxide (US-PIO) probes of 10-nm diameter are removed through ex-travasation and kidney clearance and have shorterplasma half-life times. After intravenous injectionsof clinically available USPIO (colloidal particle size15-65 nm), the particles stay in the blood until they enterthe reticuloendothelial system (macrophages of the liver,spleen, and bone marrow); a plasma half-life time of15 hours has been reported in humans. Our own data, us-ing a 10-mg/kg iron injection of commercially availableFerumoxytol (USPIO, 18-30 nm), showed that these US-PIO rapidly pass through the kidney (4-12 hours in con-trol mice) with a significant and prolonged T2-decreasein the liver (as expected because of hepatic uptake andmetabolism, Fig 2C). However, our data show that inthe inflamed kidney (a mouse ischemia/reperfusionmodel), the decrease in T2-relaxation times persistedwell over 24 hours after injection of the SPIO, indicatingmacrophage uptake of iron. Another study using approx-imately 20- to 30-nm Dextran-coated SPIO nanoparticlesexamined the contrast effect of the nanoparticles in a ratmodel of glomerular and tubulointerstitial injury.61 Ratswere injected with puromycin, and after 2 weeks they

Nanosized Contrast Agents to Detect Kidney Inflammation by MRI 495

underwent kidney imaging. The kidneys were imaged byMRI using fast low-angle shot gradient-echo sequence,and images were obtained before and 24 hours after injec-tion with the nanoparticles. The T2*-signal intensity in 4different regions of the kidney (cortex, external outer me-dulla, ‘‘deep’’ outer medulla, and inner medulla) signifi-cantly decreased after injection of the SPIO. Nosignificant changes were seen in control rats. In thepuromycin-injected rats, there was a strong correlationbetween the change in signal intensity and the numberof macrophages observed by immunohistologic analysis.

The same group applied this method in 2 additionalrat models of kidney disease.62 In one of the experimentsthe investigators induced nephrotoxic serum-mediatedglomerulonephritis in rats, a model in which inflamma-tion is restricted to the glomeruli. The authors foundthat injection with the nanoparticles caused a visibledarkening in the renal cortex as well as a significant re-duction in the T2*-signal in that region. No change inthe MRI signal was detected in the outer or inner medul-las. It is interesting to note that there was a significant de-crease in the signal intensity in the cortexes of rats within2 days of injection of the nephrotoxic serum, before infil-tration of the glomeruli with macrophages. Electron mi-croscopy demonstrated that the SPIO were presentwithin mesangial cells, and the authors posited that thiswas due to increased endocytic activity of the mesangialcells in this model. Although the endocytosis of the SPIOwas performed by mesangial cells, it only occurred afterinjection of the rats with nephrotoxic serum and stillseems to represent a signal of inflammation. On the otherhand, in a model of obstructive nephropathy, the authorsfound that injection of the rats with the nanoparticlescaused a reduction in the MRI signal in all regions ofthe kidney. On the basis of their results in differentmodels of kidney injury, the authors concluded that en-hancement of the kidneys with USPIO can be used to de-tect inflammation within the kidney and to localize themacrophage infiltrate to specific regions of the kidney.

Several studies have used SPIO nanoparticles to detecttubulointerstitial inflammation in models of AKI. Jo andcolleagues used 20- to 30-nm USPIO to detect tissue in-flammation in a rat model of ischemic AKI.63 In thesame study, the authors showed that injection of USPIOdid not cause a detectable change in the MRI signal ofrats with mercuric-chloride-induced AKI. Unfortunately,little information was given regarding the degree of kid-ney injury or macrophage infiltration in the mercuricchloride model.

Studies Using Targeted Nanoparticles

Several studies have used targeted SPIO to detect specificmolecular markers of inflammation in models of kidneydisease. Akhtar and colleagues conjugated a monoclonalantibody to mouse vascular cellular adhesion molecule 1

(VCAM-1) to the surface of 1-mm iron-oxide microparti-cles (MPIOs).64 They subjected male C57BL/6 mice to30 minutes of unilateral ischemia. After 16 to 18 hoursof reperfusion, they injected the mice with targeted or un-targetedMPIOs and obtained T2*-weighted images of thekidneys 6 times within 90 minutes of injection of theMPIOs. The VCAM-1-targeted MPIOs caused a contrasteffect in the cortex and medulla of the ischemic kidneys,and in the nonischemic kidneys to a lesser extent. Fur-thermore, the effect on the T2*-signal could be blockedby preinjecting the mice with purified antibody before in-jecting them with antibody-targeted MPIOs, confirmingthe specificity of the contrast effect.

Our group has used C3-targeted SPIO to detect kidneyinflammation in the MRL/lpr model of lupus nephri-tis.46,47 The complement protein C3 is cleaved and fixedto tissues during inflammation,65 and kidney biopsiesare routinely stained for C3 fragments. We used a re-combinant protein that incorporates the C3d bindingregion of complement-receptor-2 (CR2) to target tissue-bound C3d deposits. We conjugated the recombinantprotein to the surface of 70-nm SPIO (Fig 3). We then in-jected MRL/lpr and control mice with targeted or untar-geted SPIO and performed T2-weighted MRI of thekidneys 4, 24, 48, and 72 hours after injection of theSPIO.46 Injection of the diseased mice with the CR2-targeted SPIO caused significant negative enhancementof the kidneys. Affected regions included the cortex(Fig 4), inner medulla, and outer medulla. Injection ofcontrol animals with the targeted SPIO did not decreasethe T2-relaxation times (Fig 4), and injection of diseasedmice with untargeted SPIO did not affect the T2-intensity of the kidneys.

We next used the same method to determine whetherwe could assess disease severity in the MRL/lpr model.47

Kidney disease becomes progressively more severe as theMRL/lpr mice age, andwe confirmed that the abundanceof C3 fragments within the glomeruli increases in parallelwith the progressive worsening of disease. We imagedthe kidneys of MRL/lpr and control mice at 12, 16, 20,and 24 weeks of age. At each time point we obtainedbaseline images of kidneys and then injected the micewith CR2-targeted SPIO. Injection of the diseased micewith targeted SPIO caused negative enhancement of thekidneys by T2-weighted images. The magnitude of thischange was greatest at 20 weeks of age, the age of thegreatest abundance of glomerular C3. These studies dem-onstrate that MRI with CR2-targeted SPIO can be used toidentify immune-complex glomerulonephritis and to as-sess the severity of the disease. The biomarker of diseasedetected by the CR2-targeted SPIO (C3 activation frag-ments) is routinely examined in biopsies of patientssuspected or known to have immune-complex glomeru-lonephritis. Therefore, this molecular imaging methodcan be used to noninvasively monitor a key biomarkerthat is currently evaluated only by invasive tissue biopsy.

Figure 3. Generation of C3d-targeted SPIO nanoparticles. Iron oxide crystals can be coated with various organic and inor-ganic polymers. Targeting molecules, such as recombinant CR2, can be conjugated to the surface of the nanoparticles afterthey are coated, or they can be incorporated into the polymer before encapsulating the SPIO. This is a figurative representa-tion and does not accurately represent the scale of the final targeted SPIO. Abbreviations: CR2, complement receptor-2; SPIO,superparamagnetic iron oxide.

Thurman and Serkova496

Clinical MRI Studies on Kidney Inflammation

In current clinical practice, the degree of kidney inflamma-tion can be only determined by kidney biopsy. SPIO nano-particles are taken up by extrahepatic cells withphagocytic activity, including circulating monocytes andresident macrophages present in inflamed tissues. MRIwith iron-based nanoparticles has been used to detect kid-ney inflammation in human kidney transplant recipi-ents.48 T2*-weighted MRI was performed 72 hours afterinjection of USPIO nanoparticles. One patient (withbiopsy-proven cortical inflammation) showed a significantdecrease in T2*-signal intensity. All of the other kidney al-lograft recipients, even those with chronic and fibrotic dis-ease but with no macrophage infiltration of their biopsies,did not show any changes in T2*-signal intensity after US-PIO injection. Although this study by Hauger and col-leagues is the only study using MRI with SPIO inhuman kidneys, SPIO nanoparticles have been success-fully used in humans for detection of liver metastases; isletinflammation; lymph node MRI; and, most recently, mul-tiple sclerosis with no reported toxicities.49,66-69

Safety of Iron-Oxide Nanoparticles

Rapid infusions of iron replacement formulations cancause oxidative stress and may be damaging to the kid-neys.70,71 Toxic levels of nonchelated iron can build up,and they have the potential to produce radical oxygenspecies. However, toxic effects were not seen in rats ordogs injected with high doses (3000 mMol Fe/kg) ofSPIO.72 The reason for this is that the breakdown of mag-netite (or maghemite) in the body forms ferric (and notferrous) iron, which is then efficiently chelated byendogenous citrate, and it remains nontoxic. However,nanoparticle toxicities are potentially different for eachunique particle. Surface proteins may be immunogenic,and some surface coatings may cause anaphylaxis.

Ferumoxytol is used as an iron replacement therapyand has been administered to many patients withCKD.73 There are reports of anaphylactic reactions andhypotension to Ferumoxytol (see www.amagpharma.com/products), but the episodes are usually mild andof short duration. It is not clear whether the lower dosesof nanoparticles needed for molecular imaging studieswill pose the same risks as the higher doses used foriron replacement.

Future Directions

Although anatomical and functional imaging remain goldstandards for noninvasive assessment of kidney structureand function, recent developments in molecular MRI in-dicate that pathophysiological pathways of kidney dis-ease, including inflammation, can be visualized at thetissue level.74,75 The ultimate goal is the developmentof molecular imaging methods capable of providingclinicians with the same data that are currently providedby kidney biopsy. This would ideally include resolutionthat can approach that obtained with histologicalexamination of tissues. It would also include detection ofthe same biomarkers that are currently examined bytissues biopsies: immunoglobulins and light chains,complement proteins, inflammatory cells, fibrosis, andother deposits. SPIO-based MRI provides a promisingmethod for macrophage imaging (untargeted nanopar-ticles) and for noninvasively detecting specific molecularbiomarkers of inflammation, such as C3 fragments. For ex-ample, in the future, so-called ‘‘multifunctional’’ or ‘‘mul-timodal’’ nanoparticle probes can be applied for moresensitive detection of inflammatory target proteins usinga positron emission tomography/MRI approach.

The treatment of autoimmune and inflammatorykidney disease will improve in coming years with thedevelopment of new immunomodulatory therapies.

Figure 4. T2-mapping in the cortex of MRL/lpr mice injectedwith CR2-targeted SPIO. We have mapped T2-valuesthroughout the cortex of imaged kidneys. These T2-maps in-corporate the data of the 16 echoes used during image ac-quisition and allow the quantitative assessment of the DT2throughout a 2-dimensional image of the cortex. The dark-ening of the cortical region in 20-wk-old MRL/lpr mice (or-ange-green / dark blue) represents the decrease in theT2-time after injection with CR2-targeted SPIO. Little changeis seen in control mice after injection with the nanoparticles.Abbreviations: CR2, complement receptor-2; SPIO, super-paramagnetic iron oxide; t-SPIO, CR2-targeted SPIO. Thisanalysis was performed on images from previously pub-lished experiments.46,47 (For interpretation of references tocolor in this figure legend, the reader is referred to the webversion of this article.)

Nanosized Contrast Agents to Detect Kidney Inflammation by MRI 497

The concurrent development of molecular imagingmethods for monitoring kidney inflammation will be crit-ical for the rapid evaluation of these new therapies. Be-cause molecular imaging can be used to detectinflammatory markers throughout both kidneys, suchmethods will actually provide a much more comprehen-sive picture of kidney inflammation than a kidney biopsy.Safe methods capable of reporting the extent and distri-bution of kidney inflammation can be used to rapidly as-sess the efficacy of new anti-inflammatory therapies

without having to conduct long-term clinical studies.Such methods will also be essential for tailoring an indi-vidual patient’s treatment on the basis of their responseto therapy and the total kidney burden of inflammationand/or fibrosis.

Acknowledgments

The original studies reported in this review article were sup-ported by the University of Colorado Cancer Center P30 grantCA046934 and the Colorado Clinical and Translational SciencesInstitute UL1 award RR025780. This work was also supportedin part by the KIDNEEDS Foundation, the Lupus Research In-stitute, and the National Institutes of Health Grant R01DK076690.

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