A P P L I C AT I O N N O T E

GFR-Vivo 680
Fluorescent Imaging Agent
AuthorJeffrey D. Peterson, Ph.D.

PerkinElmer, Inc.
Waltham, MA USA

An in vivo non-invasive method todetermine glomerular filtration rate (GFR)
Abstract
Glomerular filtration rate (GFR) is the gold standard in kidney function assessment and isused to determine progression of kidney disease and drug-induced kidney toxicity. Oneof the most accepted ways to assess GFR is by measuring the rate of disappearance oflabeled inulin from the blood; as inulin is completely filtered at the kidneys’ glomeruli(but neither secreted nor reabsorbed by the tubules), this rate of disappearance isdirectly proportional to GFR. We have developed a near infrared (NIR) fluorescent-labeledform of inulin (GFR-Vivo™ 680) in a spectral region providing low background and hightissue penetration (ex/em = 670/685 nm) for in vivo application. Fluorescence molecular
tomographic (FMT) imaging of the heart was used to detect and quantify blood levelsof GFR-Vivo 680 at multiple time points, providing the necessary data to calculate theclearance rates in individual animals. Following an intravenous bolus of NIR-Inulin in
SKH-1E mice, FMT® images were acquired at 1, 5, 15, 30, and 45 minutes post-injection.
Clearance rates were calculated using a two-compartment curve fitting, yielding average
rates of 270 + 6 mL/min in normal mice. GFR-Vivo 680, in combination with FMT heart
imaging, provides a non-invasive fluorescent imaging approach to generate consistent GFR
measurements in models of kidney disease, dysfunction, and drug toxicity.

MATERIALS AND METHODS:
Fluorescent Agent

Protocol For GFR Assessment with GFR-Vivo 680

GFR-Vivo 680 is a near infrared (NIR)-labeled inulin molecule
designed for determination of glomerular filtration rate
through detection and quantification in the blood. The kinetics
of non-invasive tomographic fluorescent imaging of heart
fluorescence accurately represent the rate at which this agent isfiltered through the kidneys’ glomeruli.

Study Preparation:
1. As GFR assessment requires carefully timed injections and imaging (1,5, 15, 30, and 45 minutes) (Figure 1), it is essential to prepare aheadof time for optimal results. Group and number the mice to be injected
and imaged.

2. Mouse hair removal: Either genetically hairless mice (SKH-1E) or
normal mice (BALB/c, C57BL/6, etc.), with depilation, must be used
Pre-injection for optimal fluorescence tomographic imaging.

Basic Properties of GFR-Vivo 680
Agent Type
Molecular Weight
AbsorbanceFluorescence emission

GFR-Vivo 680
NIRF-labeled inulin~6000 g mol-1
670 nm
685 nmAgent Summary. Characteristics of the agent (MW/size, absorbance/emission)
were determined in multiple independent studies

Models of kidney dysfunction
A variety of mouse models of kidney disease, dysfunction, or
toxicity can show defined increases or decreases in glomerular
filtration rate. These include uninephrectomy and kidney remnant
models of chronic kidney disease (CKD), Type I (STZ-inducedor spontaneous NOD mouse disease) and Type II (ob/ob mice)diabetic nephropathy, drug induced kidney toxicity with a varietyof chemical agents, and infectious disease (e.g. Plasmodium
berghei, Escherichia coli). In these studies, uninephrectomized andsham-surgery SKH-1E hairless mice (Charles River Laboratories,
Wilmington, MA) were assessed seven days post-surgery as amodel to validate GFR quantification.

- To minimize light scattering and absorption in heart imaging, hairis removed from the upper torso of all mice; depilatory cream(Nair lotion, Church and Dwight Co., Inc., Princeton, NJ) is applied
thickly on skin over the upper torso (front, back, and sides) of eachmouse, rinsed off with warm water, and reapplied until all hair has
been removed.
- Care should be taken to remove hair from an area larger than justthe upper chest (i.e. including removal from mid-torso and neckregions, as well).
3. Establish the readiness of the FMT imaging system by checking
the anesthesia chamber and connections to the FMT. Activate theanesthesia, setting evaporator to 3.5.
4. TrueQuant™ software: Open FMT TrueQuant software and enter
all necessary study information as a new study. Open the image
acquisition software panel in readiness for the first mouse and selectthe first subject in the “Select Subject” field.
5. Make sure you know ahead of time the proper positioning of themouse in the imaging cassette to permit tomographic imaging of thethoracic region.

In Vivo Heart Fluorescence Imaging
Heart fluorescence is only poorly detected by epifluorescence(2D) imaging, and rapid tomographic imaging is required for the
purpose of quantifying levels of fluorescence in the heart. The
FMT 2000 or FMT 4000 fluorescence imaging systems provide
two (680 and 750 nm) or four NIR excitation lasers (635, 680,
750, and 790 nm), respectively, that are used for FMT imaging
of small animals. A NIR laser transilluminates the animal (i.e.
passes light through the body), with signal detection occurring
via a thermoelectrically cooled charge-coupled device (CCD)
camera placed on the opposite side of the imaged animal.
Appropriate optical filters allow collection of both fluorescenceand excitation datasets generated by raster-scanning themouse, and the resulting imaging dataset is used to reconstructfluorescence in 3D.

Figure 1. Kinetic Heart Imaging. Hairless or depilated mice can be imaged repeatedly
over time to quantify levels of fluorescence in the heart as a surrogate for blood measurement.
Injection of Mice with GFR-Vivo 680:
1. Prepare the GFR-Vivo 680 agent according to included instructions.
2. Place a heating pad beneath the anesthesia induction chamber tokeep the body temperature of the mouse constant. Be careful not
to overheat. Anesthetize the first mouse by placing it in the gas
anesthesia induction chamber.
3. Prepare a notebook for entry of injection times. Use the time shownon the computer monitor of the imaging system to assure alignmentof injection time with imaging times.
4. Remove the mouse from the induction chamber when it appears
completely anesthetized, and confirm the depth of anesthesia
through unresponsiveness to toe pinch.

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5. Inject 100 μL (2 nmol) of GFR-Vivo 680 via the retro-orbital plexus (ortail vein) of the anesthetized mouse. Record the time as “injection
time” or t=0.
6. Quickly position the injected mouse in the FMT’s imaging cassette(supine) and position the top plate of the cassette. Gently adjust
the cassette depth for proper and gentle restraint of the mouse(generally 13 mm depth for a 25 g mouse). This will not affect normal
respiration but will secure the mouse in place. Steps 5 and 6 shouldtake no longer than 20-30 seconds).
FMT 4000 heart imaging:
1. Insert the mouse/cassette into the FMT imaging system and close thedoor. The correct subject to be scanned should already have been
selected in the “Select Subject” field.
2. Acquire the reflectance (2D) image of the mouse and quickly size
and align the scan field to allow 45-50 scan points positioned toencompass the ribcage region (thorax) of the mouse (see Figure 2).
Select “Add to Reconstruction Queue” for automatic backgroundreconstruction of tomographic datasets.
3. Start the FMT acquisition scan to acquire the first time point scan,
initiated as close as possible to 1 minute post-injection. Record thetime that the scan is initiated. The scan will take 2-2.5 minutes.
4. As soon as the first scan is completed, turn down the evaporator tomaintenance levels (set to 1). With the lower anesthesia setting, eachmouse can be kept within the imaging chamber for the duration of
the experiment.
5. At 4 or 5 minutes post-injection, initiate the second thoracic scan.
Record the time that the scan is initiated.
6. Repeat at 15, 30, and 45 minutes. In some cases, you may want toextend the imaging to 60 minutes.
7. Note that scan initiation time can also be confirmed in the experimenttab for each animal scan (Created column).

Figure 2. TrueQuant Scan Acquisition. Following the acquisition of the reflectanceimage, a white light view of the mouse will appear with a default scan field superimposed on
the image. Resize the scan field using the computer mouse to achieve full scan coverage of theupper chest in roughly a 7 x 7 grid, or larger.

Note: Alternative overlap imaging of two mice: for maximal
imaging throughput, it is possible to image an additional
mouse during the longer imaging intervals.
a. Inject the second anesthetized mouse (described above) as
the 5 minute scan of the first mouse is completed. Record
the time for mouse 2, and replace mouse 1 in the cassettewith mouse 2.
b. Complete 1 and 5 minute imaging time points for mouse 2,
and then switch back to mouse 1 for the 15 min time point
for mouse 1.
c. Alternate the mice to complete the imaging time points forboth mice (t = 1, 5, 15, 30 and 45 min)FMT 4000 analysis:
Proper quantitative analysis of heart fluorescence by the FMT
System’s TrueQuant software requires accurate and consistentpositioning of 3D ROIs. Figure 3 shows an image of a mouseacquired on the FMT at the first time point, with an outline
to indicate the ribcage. It is important to capture heart signal
without capturing nearby liver signal (due to high vascularityof this organ). An ellipsoidal ROI is deliberately oversized tomaximize capture of all of the heart’s fluorescence. Viewed fromthe front, it is positioned roughly in the center of the ribcage,with care taken to avoid the top of the liver (the tip of which
is behind the xiphoid cartilage). All of the time point data froma single mouse can then be opened within TrueQuant in the
Analysis tab (set to 6 panels), and the ROI from the first time
point can be copied to all of the subsequent time points.

Figure 3. TrueQuant ROI Placement. A 3D ellipsoidal ROI should be placed that is
9x11x6 voxels (width x height x depth) and localized centrally in the rib cage region and set tocapture the first 6 depth voxels . Other signal will be seen due to circulation through bloodvessels in the surrounding tissues.
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No thresholding should be applied to the heart ROIs, and thepmol data will be used for subsequent calculations of bloodconcentration. This is repeated for each individual, image ultimately
yielding quantification of the complete kinetic tomographic
datasets for every animal. The complete pmol dataset is thenexported to Microsoft® Excel® for further calculations.
Calculation of blood concentration:
Although the TrueQuant software calculates the nM
concentration of fluorescence within the heart ROIs, this is an
innacurate measure of blood fluorescence, because 1) it doesn’t
account for the volume of tissue versus blood in the ROI, and2) the resolution of fluorescence tomographic reconstruction
generally oversizes the volume of fluorescence due to theinherent properties of light propagation through tissue. Thismeans that the pmol data are the most accurate readout (notsubject to these artifacts), but they need to be converted to nM
by dividing them by the blood volume within the heart.
A 25 g mouse has a blood volume of approximately 1.5 mL(see Table I). Heart fluorescence pmol are converted to nM
by making the assumption that the heart contributes 7%of the blood volume, or 0.1 mL. Based upon these reported
physiologic parameters of the mouse, the t=0 fluorescenceconcentration after injection of 2 nmol of GFR-Vivo 680 would
be 1333 nM. All imaging time point values are determined by
dividing the pmol value by 0.1 mL, yielding the concentration
within the blood of the heart in nM.
Table I. Vascular physiologic parameters in the mouse
Physiologic Parameter
Measurement in 25g Mouse
Total blood volume
1.5 mLHeart blood volume
7% of total blood volume= 0.1 mL
The imaging times are noted during the in vivo study dataacquisition, however the times should be adjusted to provide amore accurate estimate of time. As each tomographic imaging
session for a 45-50 point upper torso scan takes approximately
2 minutes, we established each time point as the scan start
time + 1 minute (the temporal midpoint of each scan). So, scan
initiations at 1, 5, 15, 30, and 45 minutes yield actual datatime points of 2, 5, 16, 31, and 46 minutes (see Table II). This
modification of times has some impact on GFR calculation but
will mostly affect calculation of blood half-life.
Table II. Calculated FMT imaging times
Imaging Start
Scan
Time (min)Duration
0
N/A
1
2 min
5
2 min
15
2 min
30
2 min
45
2 min

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Imaging Time
Point (min)
0
2
6
16
31
46

Figure 4. Kinetic heart imaging by FMT. Two representative mice (Sham andUninephrectomized) are shown at the indicated imaging times. Heart (only) signal in 3D isshown for each time point, with higher concentration signal represented by shades of red.

Calculation of glomerular filtration rate (GFR):
A two-compartment clearance model is used to calculate
GFR, providing a good fit to both the early, rapid decay phaseas well as the later, slower elimination phase. A variety of
Pharmacokinetic software (including WinNonLin [Pharsight,
Sunnyvale CA] and PK Solutions [SummitPK, Eugene OR]) canbe used to calculate pharmacokinetic parameters such as blood
half-life and clearance rates. Alternatively, GFR can also be
calculated on GraphPad Prism (GraphPad Software, San Diego,CA) using the equation:

GFR = I / (A/a + B/b)
I is the amount of inulin delivered in bolus injection; A and a are the y-intercept and decay
constant of the initial rapid decay phase, respectively; and B and b are the y-intercept and decay
constant of the slow elimination phase, respectively (Qi et al., 2004).

For our GFR studies, we used a freely available, menu-driven
add-in program for Microsoft® Excel® written in Visual Basic
for Applications (PKSolver, Zhang Yon, China Pharmaceutical
University). A detailed description and comparison by Zhang
et al, can be found in the journal Computer Methods andPrograms in Biomedicine 99 (3); 306-314 (2010). All data weretabulated in Microsoft® Excel®, and the Two Compartment IV
Bolus fit of the PKSolver Add-In was used to generate curve fits
and calculations of the clearance rates for each mouse in uL/min.
To test the imaging methodology and analysis techniques of
non-invasive GFR quantification, we imaged the effect of theremoval of one kidney (uninephrectomy) on GFR-Vivo 680
heart signal (Figure 4). Uninephrectomy should yield a delay inthe clearance of circulating fluorescence (assessed in the heart)which should translate into a 50% decrease in GFR as compared
to sham surgery mice.

Times were recorded for each Sham and Uninephrectomized
mouse, and the time points were defined relative to injection
time (t 0), taking into account the 2 minute scan time by adding
1 minute (mid-point of each scan) to each imaging time. In
Table III, data from a representative Sham surgery mouse (A)and a representative Uninephrectomized mouse (B) are shown.
The total pmols of fluorescent signal from GFR-Vivo 680 wereconverted to blood concentration (nM) by dividing by 0.1 mL(heart blood volume) as described in the previous section andin Table I. Within Excel, this dataset was analyzed using the
PKSolver Add-in with a 2-compartment curve fitting optimized
for IV bolus (Figure 5). Briefly, for each mouse the Time andConcentration datasets are selected, time unit is set in “min”,
concentration in “nmol/L”, Dose at “2”, and Dose Unit as“nmol” (Figure 6).

Table III. Converting FMT pmol to blood concentration

Curve fitting for these two representative animals fit theobserved data well and revealed a delay in apparent clearanceof GFR-Vivo 680 from circulation (Figure 7). GFR for Shamand Uninephrectomized mice were determined to be 251 and120 μL/min, respectively (Table IV), in agreement at the levelof both established normal GFR values from the literature andwith regard to the quantitative effect of uninephrectomy. Other
calculated pharmacologic parameters, including alpha- and beta
half-lives, exposure levels (AUC and AUMC), mean residence
time (MRT), and steady-state volume of distribution (Vss) allshow the expected differences between these two mice.

Figure 6. Curve Fitting with PKSolver. Data is selected for the Time andConcentration fields, and other defaults are selected as shown.

Figure 5. Curve Fit Selection with PKSolver. The PKSolver add-in, opened within
Microsoft Excel, offers many options for curve fitting of pharmacokinetic data. The
2-compartment IV bolus curve fit was used to best capture the typical kinetics of inulin
clearance.

Figure 7. Kinetic Blood Clearance Curves. Kinetic heart datasetsfrom representative Sham and Uninephrectomy mice are shown.

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In vivo GFR assay performance metrics:
Averages for normal mice and uninephrectomized mice werecompared across multiple studies. GFR-Vivo 680 assessment of
GFR by FMT tomographic imaging of the heart compared favorablywith regard to normal animals, yielding averages of 274 +/- 56 μL/
min in five studies. Uninephrectomized mice showed an average
GFR value of 132 +/- 12 μL/min in three studies.
Power analysis studies (modeled on actual variability of empiricaldata) were performed to predict the numbers of animals needed
per group to detect GFR decreases of 30-50% (to approximate
typical findings from toxicity and nephrectomy studies), and
30% to 100% increases (to approximate expected increases in
GFR expected in diabetes). Our approximations indicated thatas few as 4-5 mice per group were required to detect 2-foldchanges (either 2-fold increase or 50% decrease) and 10-15
mice were required to detect 30% changes relative to controls.
A typical blood collection study (ex vivo assessment of insulinlevels) could require from 30-60 mice, depending on institutional
IACUC rules regarding repeat blood sampling.

SUMMARY
The present studies provide evidence for the utility of GFR-Vivo
680, in combination with FMT imaging, in the non-invasive
quantification of glomerular filtration rate (GFR). Imaging andanalysis techniques are described, and the data generated
in normal and uninephrectomized mice show clear data inagreement with the range of published GFR values. Further,
GFR-Vivo 680 assessment of GFR yields an expected 50%
decrease upon removal of one kidney. Inter-animal variability
was 15-20%, mostly attributed to the variability of the biology
itself from animal-to-animal. In addition, differences would
be expected in comparison of male to female mice, mice ofdifferent ages, and in profiling different mouse strains. Looking
at 5 independent studies in SKH-1E mice, inter-assay variability
within the same mouse strain was < 10%, indicating thatthe average GFR of small groups of mice was fairly consistentdespite the animal-to-animal variability.
Statistically significant changes in GFR upon uninephrectomy
were easily detected with only 4-5 mice per group (imaged
at multiple time points), and our extrapolations predict thatas few as 10-15 mice per group would be required to detect
a 30% changes in GFR. Even increases in GFR up to 3-fold(i.e. more rapid clearance from the blood should be readily
detectable using the protocol described. In conclusion, heart
tomography of GFR-Vivo 680 fluorescence should provide arobust approach to generate consistent GFR measurements inmodels of kidney disease, dysfunction, and drug toxicity.

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Table IV. GFR and other PK measures established from FMT
heart measurement of Sham and Uninephrectomy mice

Table V. Non-invasive GFR-Vivo 680 assay
performance metrics
ResultAverage GFR in 25g female SKH-1E mice(n = 11)
Average GFR in uninephrectomized 25 g
female SKH1-E mice (n = 11)
Intra-assay variability(Coefficient of Variation)
Inter-assay variability(Coefficient of Variation)n required to detect 30% changen required to detect 2-fold change

Comment
274 ± 56 μL/min
132 ± 12 μL/min
15-20%< 10%
10-15 mice per group
4-5 mice per group

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