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STANDARD ARTICLE
Diagnostic potential of simplified methods for measuring
glomerular filtration rate to detect chronic kidney disease
in dogs
Paola Pocar
1
| Paola Scarpa
1
| Anna Berrini
1
| Petra Cagnardi
2
| Rita Rizzi
1
|
Vitaliano Borromeo
1,2
1
Department of Veterinary Medicine,
Università degli Studi di Milano, Milan, Italy
2
Department of Health, Animal Science and
Food Safety, Università degli Studi di Milano,
Milan, Italy
Correspondence
Vitaliano Borromeo, Department of Veterinary
Medicine, Via Celoria 10, 20133 Milan, Italy.
Email: vitaliano.borromeo@unimi.it
Abstract
Background: Glomerular filtration rate (GFR) is the most sensitive indicator of initial
renal function decline during chronic kidney disease (CKD), but conventional proto-
cols for measuring GFR are labor-intensive and stressful for the dog.
Objectives: To assess the diagnostic potential for detecting CKD with simplified GFR
protocols based on iohexol plasma clearance.
Animals: Seventeen CKD-positive and 23 CKD-negative dogs of different breeds
and sex.
Methods: Prospective nonrandomized study. Plasma iohexol was measured 5, 15,
60, 90, and 180 minutes after injection. Glomerular filtration rate was calculated
using 5 samples (GFR
5
) or simplified protocols based on 1, 2, or 3 samples. The GFR
5
and simplified GFR were compared by Bland-Altmann and concordance correlation
coefficient (CCC) analysis, and diagnostic accuracy for CKD by receiver operating
characteristic curves. A gray zone for each protocol was bounded by the fourth quar-
tile of the CKD-positive population (lower cutoff) and the first quartile of the CKD-
negative population (upper cutoff).
Results: All simplified protocols gave reliable GFR measurements, comparable to ref-
erence GFR
5
(CCC >0.92). Simplified protocols which included the 180-minutes sam-
pling granted the best GFR measure (CCC: 0.98), with strong diagnostic potential for
CKD (area under the receiver operating characteristic curve ± SE: 0.98 ± 0.01). A
double cutoff including a zone of CKD uncertainty guaranteed reliable diagnosis out-
side the gray area and identified borderline dogs inside it.
Conclusions: The simplified GFR protocols offer an accurate, hands-on tool for CKD
diagnosis in dogs. The gray zone might help decision-making in the management of
early kidney dysfunction.
Abbreviations: AUC, area under the curve; AURC, area under the ROC curve; BSA, body surface area; BW, body weight; CCC, concordance correlation coefficient; CKD, chronic kidney disease;
CKD−, CKD negative; CKD+, CKD positive; CV, coefficient of variation; estVd, estimated Vd; GFR, glomerular filtration rate; GFR
1
, single-sample GFR; GFR
2
, 2-sample GFR; GFR
3
, 3-sample
GFR; GFR
5
, 5-sample GFR; HPLC-UV, high-performance liquid chromatography-ultraviolet; IRIS, International Renal Interest Society; ROC, receiver operating characteristic; RS-GFR, reduced-
sampling GFR; SCr, serum creatinine; SS-GFR, single-sampling GFR; Vd, volume of distribution.
Received: 16 November 2018 Accepted: 11 July 2019
DOI: 10.1111/jvim.15573
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for commercial purposes.
© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.
J Vet Intern Med. 2019;33:2105–2116. wileyonlinelibrary.com/journal/jvim 2105
KEYWORDS
gray zone approach, HPLC, Iohexol, limited sampling method, single sample method
1|INTRODUCTION
Chronic kidney disease (CKD) is an irreversible, progressive deteriora-
tion of renal function, with a poor prognosis.
1
Dogs with CKD can be
classified in stages according to the International Renal Interest Soci-
ety (IRIS) system, which is based on the concentration of serum creati-
nine (SCr), with substaging based on the urinary protein/urinary
creatinine ratio and blood pressure. However, SCr sensitivity is low
and shows alterations only after two-thirds of functional renal mass
have been lost, so it cannot detect early kidney dysfunction.
2,3
Glo-
merular filtration rate (GFR) is currently considered the best indicator
of renal function and the most sensitive and specific test for early
diagnosis of CKD.
4,5
In veterinary practice, a routine method for measuring GFR is based
on monitoring plasma clearance of iohexol by high-performance liquid
chromatography-ultraviolet (HPLC-UV).
6-9
One major limitation is that
conventional iohexol clearance protocols require repeated blood sam-
pling over several hours, which is labor-intensive, time-consuming, and
stressful for the animal. Numerous studies have attempted to deter-
mine the smallest number of blood samples needed for accurate GFR
measurements in dogs and cats,
10-12
and single-point GFR methods
have been investigated.
13-17
However, most of these methods were
not considered sufficiently reliable for CKD diagnosis.
Chronic kidney disease is a progressive disease, with a gradual
decline in GFR. Currently, for CKD diagnosis, the GFR cutoff, giving
the best compromise between sensitivity and specificity, is usually
selected.
15,18,19
However, this approach transforms the GFR into an
artificially “black or white”statistical index and can easily lead to mis-
classification of borderline cases. To tackle this problem in humans, a
3-zone partition based on 2 cutoffs including a middle “gray zone”of
uncertain diagnosis has been proposed.
20,21
The GFR gray zone
approach for CKD diagnosis has not yet been investigated in veteri-
nary clinical practice.
The primary aim of the present study was to assess the reliability
of a panel of simplified iohexol plasma clearance protocols to measure
GFR in dogs and investigate the application of a gray-zone strategy to
identify subjects at risk of CKD.
There is debate in the scientific community about the best way
to calculate the iohexol concentration in plasma by HPLC-UV and to
normalize clearance to canine body size. High-performance liquid
chromatography-UV detects 2 separate iohexol isomers: exo- and
endo-iohexol, and discrepancies have been reported by calculating GFR
based on the plasma clearance of the isomers separately
6,8,19,22,23
or
the total iohexol.
4
In addition, differences have been reported between
estimated levels of renal function standardized to body size (body
weight [BW] versus body surface area [BSA]).
4,11,15,24
Therefore, the
secondary aim of this study was to investigate which method to
compute iohexol plasma concentration and standardize GFR to canine
body size fitted best in our clinical settings.
2|MATERIALS AND METHODS
2.1 |Animals
Forty privately owned dogs of different breeds and sex were included.
Body weights ranged from 3.9 to 46.0 kg (mean ± SD, 25.1 ± 9.7 kg).
The dogs were aged 6 months-16 years (mean ± SD, 5.4 ± 3.5 years).
None of them received medical treatment before the GFR assessment,
and they were fed their usual food, with water ad libitum. Each dog had
a complete physical examination shortly before GFR was measured.
One blood and 1 urine sample were collected before the iohexol injec-
tion, for CBC, serum biochemistry profile, and routine urinalysis. Exclu-
sion criteria included abnormal diagnostic screening test results or dogs
receiving medications. Healthy was defined as the absence of any clini-
cal signs or relevant abnormalities on physical examination, CBC, serum
biochemistry profile, routine urinalysis, and ultrasound examination.
Chronic kidney disease was assessed according to the IRIS guidelines.
25
Healthy dogs and IRIS stage 0 were considered CKD-negative (CKD−,
23 dogs) and those with CKD IRIS stages 1 or higher CKD−positive
(CKD+, 17 dogs).
2.2 |Iohexol injection and blood sampling
The protocol was based on Lippi et al
26
with some modifications.
Briefly, food was withheld from each dog for at least 12 hours before
the procedure. Dogs were allowed free access to water throughout
the study. Dogs were weighed, and indwelling catheters were placed
in the right and left cephalic veins. A commercially available iohexol
formulation (Omnipaque; Nycomed Amersham Sorin, Milan, Italy) was
used. The nominal dose of iohexol was 64.7 mg/kg, and the exact
dose was determined from the difference between the weights of the
syringe before and after the injection. Iohexol was injected as a
60-second IV bolus into the catheter in the left cephalic vein. Two mil-
liliters of blood were directly sampled from the right cephalic vein,
transferred to a heparinized tube, and centrifuged. Samples were
immediately centrifuged at 2000gfor 15 minutes, and plasma was
stored at −30C until use. Samples were taken 5, 15, 60, 90, and
180 minutes after injection of the marker.
2.3 |Iohexol HPLC measurements
Iohexol was determined using a Waters 626 HPLC system with a
996 photodiode array detector (Waters, Milford, Massachusetts) (1spec-
trum/second; wavelength 200-320 nm, extracting the chromatogram at
254 nm). Iohexol was separated in a Simmetry100 C18 column, 3.5 μm,
2106 POCAR ET AL.
2.1 x 150 mm (Waters) using a mixture of CH
3
CN and 0.1%
orthophosphoric acid in water (3:97, vol/vol) at a flow rate of
0.3 mL/min. During separation, the column was held at 30C. Standard
iohexol (Omnipaque 350; 755 mg/mL iohexol) was added to untreated
dog plasma to obtain the following standard solutions: 5, 20, 50, 200,
and 500 μg iohexol/mL. Plasma samples were deproteinized with 5%
perchloric acid (1:1, vol/vol), centrifuged at 11000 gfor 10 minutes at
5C, and 10 μL of supernatants were injected into the HPLC column.
Data was processed using Millennium software (Waters). The peak areas
of both iohexol isomers were used to calculate the iohexol concentra-
tions and plasma clearance. The long-term stability of iohexol in plasma
was tested by reanalysis after 36 months in a freezer at −30C, on
60 samples collected during the GFR
5
test of 12 dogs.
2.4 |Calculation of GFR: multisample methods
Multisample GFR was determined by calculating the rate of iohexol
clearance using Phoenix WinNonlin software (version 8.0; Certara L.P.,
St. Louis, Missouri). Plasma clearance was determined with the follow-
ing formula
Clearance =dose of iohexol injected
AUC ,
where AUC is the area under the curve calculated from plasma iohexol
disappearance curves after an IV bolus.
Reference GFR values (GFR
5
) were calculated by plotting the
iohexol concentration against the sampling time for 5 samples (5, 15,
60, 90, and 180 minutes after iohexol), and AUC was calculated by
the trapezoidal method with a non-compartmental pharmacokinetic
model (linear log trapezoidal with extrapolation to infinity).
To calculate GFR with reduced sampling (RS-GFR), AUC was cal-
culated using a 1-compartment model during the mono-exponential
time-part of the curve, defined by samples collected at 60, 90, and
180 minutes. The missing area due to the early fast drop of the
disappearance curve was corrected by a current dog formula for
1-compartment assumption, according to Heiene et al.
27
Reduced-sampling GFR were calculated either with 3 blood sam-
ples (GFR
3
: 60, 90, and 180 minutes after injection) or a combination of
2 sampling times (GFR
2
60-90, GFR
2
90-180, and GFR
2
60-180). Samples
taken before 60 minutes were not used for 1-compartment estimates
because the terminal mono-exponential slope was often not reached
before the 1-hour sample.
Clearance (mL/min) was normalized to BW and BSA (0.101 x [BW in
kg]
0.71
) to obtain GFR, which was expressed as mL/min/kg or
mL/min/m
2
,respectively.
2.5 |Calculation of GFR: single-sample methods
The iohexol concentrations in blood samples collected at 60, 90, or
180 minutes were used to derive the equations to predict GFR for the
single-sampling protocol (SS-GFR: GFR
1
60, GFR
1
90, and GFR
1
180).
We followed a 3-step procedure, previously described.
4,16
The proce-
dure is based on the following Jacobsson formula:
GFR =1
t
Vd +0:0016
×ln Dose
Vd ×Ct,
where Vd is the volume of distribution (mL) at sample collection time
t(min), Ct the iohexol concentration measured at t, and dose is the
amount of iohexol injected for each dog (mg/kg).
First, the iohexol Vd at 60, 90 and 180 minutes (Vd
60
,Vd
90
, and
Vd
180
) for individual dogs were calculated by substituting the refer-
ence GFR
5
calculated as described in the previous section and the
plasma iohexol concentrations (Ct) at 60, 90, or 180 minutes into the
Jacobson formula and solving the formula with the “Goal-Seek”com-
mand of Microsoft Office Excel (Microsoft 2007, Microsoft Co.).
Second, the Vd
60
,Vd
90
, and Vd
180
and the plasma iohexol concen-
trations at 60, 90, or 180 minutes for each of the 40 dogs were plot-
ted in scatter diagrams, and 3 exponential equations fitting the data
were calculated, as follows:
estVDt=C0e−bCt,
where C
0
is the estimated plasma iohexol concentration at time 0; C
t
is the plasma iohexol concentration 60, 90, or 180 minutes after injec-
tion; bis the elimination rate constant, and eis the base of the natural
logarithm. These equations were used to calculate an estimated Vd
(estVd) in each dog from the iohexol concentrations found in single
samples collected at 60 minutes (estVd
60
), 90 minutes (estVd
90
), or
180 minutes (estVd
180
).
Third, the estVd
60
, estVd
90
, and estVd
180
and the iohexol dose
injected in individual dogs were put back into the Jacobsson formula
to obtain GFR
1
60, GFR
1
90, and GFR
1
180.
2.6 |Validation data set—Testing the estVd
180
formula
The estVd
180
formula determined using the 40 dogs (training data set)
was validated in an independent group of dogs (validation data set).
Clinical examinations and GFR
5
protocol were the same as for the
training data set dogs. The validation data set consisted of 11 client-
owned dogs, aged 2-14 years (mean ± SD: 6.6 ± 3.2 years), with BWs
from 9.2 to 40.3 kg (mean ± SD, 25.4 ± 10.7 kg). Seven dogs were
CKD−and 4 CKD+. Estimated Vd
180
(estVd
180val
) was calculated by
inserting the iohexol concentration at 180 minutes for each dog into
the estVd
180
formula, and GFR (GFR
1
180
val
) was calculated by
substituting estVd
180val
into the Jacobson formula. The GFR
1
180
val
and the reference GFR
5
(GFR
5val
) were then compared.
2.7 |Data analysis
Statistical analyses were done using Graphpad Prism 5.0 (Graphpad
Software, San Diego, California) and MedCalc 18 (MedCalc Software,
Mariakerke, Belgium). Differences between iohexol isomer peak areas
POCAR ET AL.2107
in fresh and stored plasma samples were analyzed using the paired
Student's ttest. Glomerular filtration rate absolute values for all
40 dogs, measured with each of the methods, were compared by
repeated-measure analysis of variance followed by post hoc Tukey's
test. Significance was set at P≤.05.
Agreement between simplified GFR protocols and reference GFR
5
was calculated using Lin's concordance correlation coefficient (CCC)
as an indicator of the degree to which paired observations fell on the
line of identity.
28
According to McBride
29
CCC >0.99, 0.95, 0.90, and
<0.90 were defined as almost perfect, substantial, moderate, and poor
degrees of agreement between methods, respectively. The agreement
was further checked graphically by plotting the difference between
FIGURE 2 Representative plasma profiles of iohexol
concentrations (mean ± SEM) in 3 dogs with high (white circles) and
low (black circles) glomerular filtration rate after an IV bolus. Time
0 was designated as time of injection. A, Arithmetic plot of iohexol
plasma concentration versus time. B Semilogarithmic plot of the same
plasma profiles limited to the iohexol elimination phase (60-180)
050 100 15 0
0
10
20
30
40
50
Clearance (mL/min)
BW (kg)
1. 5 2.0 2.5 3.0 3.5 4.0
0
10
20
30
40
50
GFR5 (mL/min/kg)
BW (kg)
050 100 15 0
0.0
0.5
1. 0
1. 5
2.0
Clearance (mL/min)
BSA (m2)
20 40 60 80
0.0
0.5
1. 0
1. 5
2.0
GFR5 (mL/min/m2)
BSA (m2)
(A)
(B)
(C)
(D)
FIGURE 3 Effect of standardizing iohexol clearance to body
weight (BW) or body surface area (BSA). Clearances were calculated
using the 5-sample protocol in 23 dogs negative to chronic kidney
disease. A, Correlation between clearance and BW. B, Correlation
between glomerular filtration rate (GFR) expressed as mL/min/kg and
BW. C, Correlation between clearance and BSA. D, Correlation
between GFR expressed as mL/min/m
2
and BSA
0.38
0.34
0.30
0.26
0.22
0.18
0.14
0.10
0.06
0.02
-0.02
AU
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Minutes
Exo – 6.184
Endo – 5.443
FIGURE 1 HPLC-UV chromatogram of a deproteinized plasma
sample from a representative dog after intravenous iohexol
2108 POCAR ET AL.
GFR
5
and the GFR values derived by each simplified protocol against
the average of the 2 values for each dog (Bland-Altman plots). Bias
was defined as the group mean difference between 2 GFR values, and
the absolute limits of agreement were defined as the group mean dif-
ference ± 1.96 SD. Data were analyzed on unit differences and per-
centage differences plots.
To determine the diagnostic consistency for CKD diagnosis of
the different GFR methods, we analyzed receiver operating charac-
teristic (ROC) curves. The calculated sensitivity was plotted against
100-specificity for different cutoff points, and the area under the
ROC curve (AURC) was used to compare the sampling protocols. The
overall accuracy of the different methods was compared according to
DeLong
30
with Pvalues considered significant at P<.05.
To establish the GFR gray zone of diagnostic uncertainty for CKD,
2 cutoff points were identified for each GFR protocol. The gray-zone bor-
ders were identified as the fourth quartile of the CKD+ population (lower
cutoff) and the first quartile of the CKD−population (upper cutoff).
3|RESULTS
3.1 |Iohexol measured by HPLC
The HPLC conditions provided good peak shapes (Figure 1), with the
stereoisomers eluting at 5.44 and 6.18 minutes for endo-iohexol and
exo-iohexol, respectively. The total run was accomplished in 21 minutes,
including equilibration of the column. The specificity of the method was
tested by analyzing plasma samples before the iohexol injection. No
interfering peaks were observed at the elution times as iohexol isomers.
The limit of quantification was 1.80 μg/mL, and the assay was linear
over the concentration range of 5-500 μg/mL, with an average
regression coefficient of 0.99 (n = 22). For all calibration curves, the
y-intercepts were virtually zero, indicating the absence of endogenous
interferences. Precision, expressed as inter-day coefficient of variation
(CV%), ranged from 4.4% to 7.8% and the intra-day CV% from 3.2% to
5.9%. Accuracy ranged from 92% to 116%.
In the Omnipaque solution, the mean ratio of the isomer peak areas
(calculated at different iohexol concentrations) was 18.99 ± 0.98 for
endo-iohexol and 81.04 ± 0.96 for exo-iohexol. The ratio was the same
in all plasma samples after iohexol injection analyzed within 2 months
from collection (P≤.001). However, in plasma samples stored for lon-
ger (36 months at −30C), about 5% of the endo-iohexol peak area was
shifted to the exo-iohexol peak area, independently from the iohexol
concentration. In the same samples before and after storage, the
mean ratios were 20.23 ± 0.83 and 15.83 ± 0.79 for endo-iohexol,
and 79.77 ± 0.83 and 84.17 ± 0.80 for exo-iohexol (mean ± SD; P≤.001;
60). The combined isomer area did not change, independently from the
iohexol concentration (P≤.001). We therefore used the combined peak
area of the 2 isomers for the quantification of iohexol in plasma.
3.2 |GFR
5
and relation to body size
No adverse clinical signs were observed during or after the injection of
iohexol in any dog. The GFR was first assessed using the reference
GFR
5
method, based on the iohexol concentrations measured during
the distribution and elimination phases, using the plasma samples col-
lected 5, 15, 60, 90, and 180 minutes after injection. Figure 2A shows a
representative example of the curves for plasma iohexol concentrations
plotted against time in dogs with high and low GFR. The 60-minute
sample was considered the starting point of the elimination phase, on
the basis of the linearity of the semilogarithmic plot of plasma iohexol
concentrations against time for the last 3 samples (Figure 2B).
The GFR
5
in the 23 CKD−dogs were used to establish the best
method for standardizing the GFR to body size. The strong correlation
between unscaled clearance and BSA and BW (Pearson's r, 0.91, for
clearance against BW and also for clearance against BSA; P≤.001;
Figure 3A) was lost when clearance was standardized to kg (Pearson's
r, 0.13; P= .58), but not when scaled to m
2
(Pearson's r, 0.66;
P≤.001) (Figure 3B). In our setting, normalization to BW was in fact
the better method, so GFR was expressed as min/mL/kg. The descrip-
tive statistic for GFR
5
indexed to BW in the 40 dogs is reported in
Table 1.
3.3 |GFR
3
and GFR
2
Glomerular filtration rate in the 40 dogs was measured using
methods based on a reduced number of blood samples. In
TABLE 1 Descriptive statistics of the
GFR measured by different protocols in
40 dogs
Minimum Maximum Mean Median IQR
GFR
5
0.25 3.43 2.12 2.15 1.42-2.80
GFR
3
0.18 3.76 2.12 2.13 1.36-2.78
GFR
2
60-90 0.15 3.72 2.14 2.20 1.50-2.78
GFR
2
60-180 0.16 3.83 2.13 2.14 1.38-2.81
GFR
2
90-180 0.20 3.65 2.12 2.17 1.39-2.79
GFR
1
60 0.82 3.99 2.09 2.14 1.31-2.60
GFR
1
90 0.76 4.04 2.19 2.27 1.40-2.77
GFR
1
180 0.52 4.03 2.16 2.14 1.29-2.86
Values are expressed as mL/min/kg.
Abbreviations: GFR, glomerular filtration rate; GFR
1
, single-sample GFR protocol; GFR
2
, 2-sample GFR
protocol; GFR
3
, 3-sample GFR protocol; GFR
5
, 5-sample GFR protocol; IQR, interquartile range.
POCAR ET AL.2109
1 there were 3 samples (GFR
3
, 60-90-180 minutes), and
3 methods used 2 samples, in all the possible time combina-
tions during the elimination phase (GFR
2
, 60-90, 90-180, and
60-180 minutes). The descriptive statistics for GFR
3
,GFR
2
60-90,
GFR
2
90-180, and GFR
2
60-180 are shown in Table 1. None of the dif-
ferences were significant.
Agreement between GFR
5
and RS-GFR was investigated using
Bland-Altman plots (Figure 4; Table 2). Biases were close to 0 with
the line of equality lying within the confidence interval of the bias,
with narrow limits of agreement. Bias values for all 4 methods were
constant throughout the range of GFR, both as absolute numbers
and percentages. Lin's CCC between GFR
5
and RS-GFR indicated
substantial agreement for all methods except GFR
2
60-90, for which
agreement was moderate (Table 2). Based on the CCC, agree-
ment with GFR
5
followed the order GFR
2
60-180 > GFR
3
>
GFR
2
90-180 > GFR
2
60-90.
3.4 |The single-sample GFR
Using the GFR
5
measured in the 40 dogs, the following formulae for
the estVd at a desired time (eg, 60, 90, and 180 minutes) were derived
from scatter plots (Figure 5).
estVD60 = 514:9e−0:014CestVD90 = 499:4e−0:013CestVD180 = 309:1e−0:01C
:
From these equations, SS-GFR was back-calculated for each dog
at 3 time points (GFR
1
60, GFR
1
90, and GFR
1
180). The descriptive
statistic for these 3 GFR
1
isshowninTable1.Nonewere
significant.
The SS-GFRs were compared to the reference GFR
5
. Bland-
Altman plots showed narrow limits of agreement, biases very
close to 0 and consistent across the range of values (Table 3;
Figure 6). The line of equality lay within the confidence interval of
the bias for GFR
1
60 and GFR
1
180 but not for GFR
1
90. Lin's CCC
between GFR
5
and SS-GFR indicated substantial agreement for
GFR
1
180 and moderate agreement for GFR
1
60, GFR
1
90. On the
basis of the CCC, agreement within protocols followed the order
GFR
1
180>GFR
1
90>GFR
1
60.
3.5 |Testing the estVd
180
formula
In the 11 dogs of the validation data set, the GFR
1
180
val
ranged
from 0.60 to 3.01 mL/min/kg (mean ± SD: 1.90 ± 0.88) and the
GFR
5val
from 0.57-3.01 mL/min/kg (mean ± SD: 1.92 ± 0.86). Agree-
ment between GFR
1
180
val
and reference GFR
5val
was evaluated by
CCC and Bland-Altman plots (Figure 7; Table 4). Lin's CCC indicated
substantial agreement (CCC: 0.98). Bland-Altmann analysis indicated
biases close to 0 with the line of equality lying within the confidence
interval of the bias, with narrow limits of agreement. Bias values
were constant throughout the range of GFR, both as absolute num-
bers and percentages.
3.6 |The diagnostic potential of the different GFR
methods for CKD
We tested the diagnostic performances for CKD of GFR
5
, GFR
3
,
GFR
2
60-180, GFR
2
90-180, and GFR
1
180 based on evidence of the
0 1 2 3 4
-0.6
-0.3
0.0
0.3
0.6
Mean of GFR5 and GFR3
0 1 2 3 4
-0.6
-0.3
0.0
0.3
0.6
Mean of GFR5 and GFR2 60-90
0 1 2 3 4
-0.6
-0.3
0.0
0.3
0.6
Mean of GFR5 and GFR2 90-180
0 1 2 3 4
-0.6
-0.3
0.0
0.3
0.6
Mean of GFR5 and GFR2 60-180
GFR2 60-180 - GFR5GFR2 90-180 - GFR5GFR2 60-90 - GFR5GFR3 - GFR5
FIGURE 4 Bland-Altman plots illustrating agreement between
reduced sampling methods (GFR
3
and GFR
2
) and the multisampling
reference protocol (GFR
5
). Differences are expressed as absolute
values. The bold line indicates the bias and the dashed lines indicate
95% upper and lower limits of agreement (mean difference ± 1.96
SD). The gray area illustrates the confidence interval of the mean
difference. GFR, glomerular filtration rate
2110 POCAR ET AL.
best agreement of these methods with the reference GFR
5
protocol
as shown by CCC ≥0.95.
Descriptive statistics for GFR in CKD+ and CKD−dogs with the
selected protocols are reported in Table 5. The AURC for each of
these protocols was ≥0.98 (Table 6), indicating strong diagnostic
potential for CKD. The AURC did not differ significantly for the differ-
ent methods.
Table 7 shows the GFR cutoffs employed to define gray zones
of diagnostic uncertainty for CKD in different GFR protocols.
Figure 8 shows the distribution of the GFR measured by the vari-
ous protocols in the 17 CKD+ and 23 CKD−dogs in relation to
the respective gray zones. Classification of the 40 dogs was con-
sistent between protocols, despite some differences in the gray-
zone limits and width. For each protocol, 13 dogs were correctly
classified as CKD+ and 18 dogs were correctly classified as CKD
−. Nine lay within the gray zone, of which 5 were CKD−and
4CKD+.
4|DISCUSSION
We found that CKD in dogs can be diagnosed with satisfactory accu-
racy using GFR calculated from a limited number of blood samples—
from 1 to 3—with flexible sampling schedules.
TABLE 2 Agreement between reduced
sampling GFR methods (GFR
3
and GFR
2
)
for measuring GFR and the multisampling
GFR reference protocol (GFR
5
)
GFR
3
GFR
2
60-90 GFR
2
90-180 GFR
2
60-180
Concordance correlation coefficient 0.98 0.94 0.98 0.98
Bias ± SD (mL/min/kg) 0.002 ± 0.16 0.02 ± 0.20 0.006 ± 0.17 0.01 ± 0.17
95% lower/upper LoA (mL/min/kg) −0.30/0.31 −0.37/0.41 −0.33/0.35 −0.32/0.34
Bias ± SD (%) −1.88 ± 9.84 0.52 ± 12.92 −1.64 ± 20.25 1.99 ± 11.68
95% lower and upper LoA (%) −21.17/17.41 −25.84/24.80 −21.72/18.45 −24.89/20.90
Abbreviations: GFR, glomerular filtration rate; GFR
2
, 2-sample GFR protocol; GFR
3
, 3-sample GFR
protocol; GFR
5
, 5-sample GFR protocol; LoA, Limits of agreement.
FIGURE 5 Scatter plots of estimated volumes of distribution
(estVd) and plasma iohexol concentrations (Ct) 60 minutes (A),
90 minutes (B), and 180 minutes (C) after bolus iohexol injection in
40 dogs. Solid lines indicate exponential trend
TABLE 3 Agreement between single-sampling GFR methods
(GFR
1
) for measuring GFR and the multisampling GFR reference
protocol (GFR
5
)
GFR
1
60 GFR
1
90 GFR
1
180
Concordance correlation
coefficient
0.92 0.94 0.95
Bias ± SD (mL/min/kg) −0.08 ± 0.31 0.10 ± 0.25 0.08 ± 0.27
95% lower and upper
LoA (mL/min/kg)
−0.70/0.53 −0.40/0.60 −0.45/0.61
Bias ± SD (%) −3.40 ± 27.35 5.87 ± 20.65 2.94 ± 15.45
95% lower and
upper LoA (%)
57.00/50.20 34.60/46.34 −27.34/33.22
Abbreviations: GFR, glomerular filtration rate; GFR
1
, single-sample GFR
protocol; GFR5, 5-sample protocol; LoA, Limits of agreement.
POCAR ET AL.2111
The GFR measured by the RS- and SS-protocols proved reliable
for many clinical situations regardless of the level of renal function.
This is in agreement with previous reports that GFR assessed by sim-
plified sampling approaches in companion animals correlates with
GFR based on multi-sample investigations.
13,16,22,27
There might be several reasons for the overall agreement we
found between the GFR
5
and the various RS- and SS-GFR. First, to
compute the simplified GFR, we used only samples collected during
the terminal mono-exponential phase. This is essential when using
simplified protocols for GFR so as to avoid loss of accuracy.
4,8,24
The
timing we considered—as the end of the iohexol distribution phase (ie,
60 minutes after the injection)—agrees with previous reports and with
the average half-life of iohexol.
8,31
Second, the RS-GFR was calculated employing a dog-specific
1-compartment correction formula according to Heiene et al.
27
Third,
to calculate the SS-GFR, we derived the reference regression curves
for Vd estimation using data from dogs with a wide range of GFR. This
might be essential to ensure reliable estimates of GFR from single
blood samples.
10
The GFR given by the simplified protocols did not significantly dif-
fer from GFR
5
, but concordance was best when the calculation
included the sample collected 180 minutes after iohexol injection, as
indicated by higher CCC and lower biases. This implies that for the
best performance, we can reduce the number of blood samples but
not the time needed for the clearance test (3 hours). This too is in
agreement with previous studies,
13,19
and the rationale is that the
timing of the last sample determines the percentage of AUC extrapo-
lated to infinity by the pharmacokinetic model compared to total
AUC. The larger this proportion, the less accurate the clearance esti-
mate.
12
Furthermore, for SS-GFR methods based on Jacobsson's for-
mula, if the sampling does not extend to late enough times after
injection, the GFR can be overestimated, especially for lower rates.
13
The simplified GFR protocols that proved most reliable (GFR
3
,
GFR
2
90-180, GFR
2
60-180 and GFR
1
180) were then examined for
their diagnostic power. The four methods showed strong potential to
0 1 2 3 4
-1.0
-0.5
0.0
0.5
1. 0
Mean of GFR5 and GFR1180
Mean of GFR5 and GFR190
Mean of GFR5 and GFR160
GFR1180 - GFR5
GFR190 - GFR5
GFR160 - GFR5
0 1 2 3 4
-1.0
-0.5
0.0
0.5
1. 0
0 1 2 3 4
-1.0
-0.5
0.0
0.5
1. 0
FIGURE 6 Bland-Altman plots illustrating agreement between
single-sample methods (GFR
1
) and the multi-sampling reference
protocol (GFR
5
). Differences are expressed as absolute values. The
bold line indicates the bias and the dashed lines indicate 95% upper
and lower limits of agreement (mean difference ± 1.96 SD). The gray
area illustrates the confidence interval of the mean difference. GFR,
glomerular filtration rate
FIGURE 7 Bland-Altman plot of single-sample method
(GFR
1
180
val
) and the multi-sampling reference protocol (GFR
5val
)in
the validation data set. Differences are expressed as absolute values.
The bold line indicates the bias and the dashed lines indicate the 95%
upper and lower limits of agreement (mean difference ± 1.96 SD). The
gray area illustrates the confidence interval of the mean difference.
GFR, glomerular filtration rate
TABLE 4 Agreement between the GFR
1
180
val
and the
multisampling GFR reference protocol in the validation data set
GFR
5val
versus GFR
1
180
val
Concordance correlation coefficient 0.98
Bias ± SD (mL/min/kg) −0.01 ± 0.16
95% lower and upper LoA (mL/min/kg) −0.33/0.31
Bias ± SD (%) 0.80 ± 8.42
95% lower and upper LoA (%) −15.71/17.30
Abbreviations: GFR, glomerular filtration rate; GFR
1
, single-sample GFR
protocol; GFR5, 5-sample protocol; LoA, Limits of agreement.
2112 POCAR ET AL.
classify CKD+ and CKD−dogs (AURC >0.98).
32
The use of a single
cutoff can easily lead to misclassification of borderline cases, espe-
cially in the diagnosis of progressive diseases like CKD. Therefore, we
tested the concept of a gray zone, identifying an interval where the
GFR gave uncertainty about the CKD diagnosis.
Different approaches can be used to establish the cutoffs for a
gray-zone.
21,33
Glomerular filtration rate in dogs is variable, with intra-
individual and interindividual CV up to 20%.
34
According to Hazra and
Gogtay,
35
when there are wide differences, it is appropriate to use a
quartile range to establish reference ranges for a defined population.
We therefore defined a fairly wide GFR gray zone, spanning from the
fourth quartile of the diseased dogs (ie, CDK+ dogs with the highest
GFRs) to the first quartile of the healthy dogs (ie, CDK−dogs with the
lowest GFRs). This achieved not only 100% specificity and sensitivity
for CKD diagnosis outside the gray zone but also permitted consistent
classification of the dogs, independent of which protocol was used for
GFR measurement.
Several studies have found that moving away from the dichoto-
mous division of quantitative test scales and identifying intermediate
range(s) of test results gave a better understanding of the diagnostic
accuracy potential of a test.
36
We therefore suggest that in veterinary
clinical practice, this approach—which clearly establishes the lower
and upper thresholds—should facilitate clinical decisions. A GFR falling
in the gray zone would not be totally uninformative as it could lead
TABLE 5 Descriptive statistics of GFR measured with different protocols in CKD+ and CKD−dogs
Minimum Maximum Mean Median IQR
GFR
5
CKD−1.98 3.43 2.65 2.69 2.18-2.99
CKD+ 0.25 2.02 1.34 1.38 1.12-1.69
GFR
3
CKD−2.06 3.76 2.68 2.73 2.46-2.94
CKD+ 0.18 2.08 1.30 1.33 0.99-1.77
GFR
2
60-180
CKD−2.06 3.83 2.70 2.68 2.47-3.00
CKD+ 0.16 2.07 1.30 1.35 0.99-1.80
GFR
2
90-180
CKD−2.07 3.65 2.69 2.66 2.41-2.93
CKD+ 0.20 2.12 1.30 1.32 0.99-1.76
GFR
1
180
CKD−1.90 4.03 2.81 2.79 2.33-3.09
CKD+ 0.52 2.05 1.25 1.25 0.99-1.44
Values are expressed as mL/min/kg.
Abbreviations: CKD, chronic kidney disease; CKD−, CKD-negative dogs; CKD+, CKD-positive dogs; GFR, glomerular filtration rate; GFR
1
, single-sample
GFR protocol; GFR
2
, 2-sample GFR protocol; GFR
3
, 3-sample GFR protocol; GFR
5
, 5-sample GFR protocol.
TABLE 6 ROC curve analysis for
chronic kidney disease identification with
different GFR measurement protocols
GFR
5
GFR
3
GFR
2
60-180 GFR
2
90-180 GFR
1
180
AURC ± SE 0.99 ± 0.007 0.99 ± 0.004 0.99 ± 0.003 0.99 ± 0.007 0.98 ± 0.01
(95% CI) (0.90 to 1.00) (0.91 to 1.00) (0.91 to 1.00) (0.90 to 1.00) (0.87 to 1.00)
Abbreviations: AURC, area under the ROC curve; CI, confidence intervals; GFR, glomerular filtration rate;
GFR
1
, single-sample GFR protocol; GFR
2
, 2-sample GFR protocol; GFR
3
, 3-sample GFR protocol; GFR
5
,
5-sample GFR protocol; ROC, receiver operating characteristic.
TABLE 7 GFR cutoffs defining the
gray zone of diagnostic uncertainty for
chronic kidney disease in different GFR
measurement protocols
GFR
5
GFR
3
GFR
2
60-180 GFR
2
90-180 GFR
1
180
Lower cutoff 1.69 1.77 1.80 1.76 1.44
95% CI 1.40-1.99 1.34-1.97 1.36-2.02 1.38-1.93 1.29-1.94
Upper cutoff 2.18 2.46 2.47 2.41 2.33
95% CI 2.12-2.50 2.12-2.52 2.13-2.51 2.15-2.54 2.17-2.67
Gray zone width 0.49 0.69 0.67 0.65 0.89
Values are expressed as mL/min/kg.
Abbreviations: CI, confidence intervals; GFR, glomerular filtration rate; GFR
1
, single-sample GFR
protocol; GFR
2
, 2-sample GFR protocol; GFR
3
, 3-sample GFR protocol; GFR
5
, 5-sample GFR protocol.
POCAR ET AL.2113
the veterinarian to seek further evidence of kidney failure and, if nec-
essary, to adopt measures to slow its progression.
We are aware that simplifying sampling method increases the
chance of errors in the estimation of GFR, especially single-sample
methods because of their empirical character. Indeed, among the GFR
protocols based on a limited number of blood samples, GFR
1
were
those with the lowest agreement with GFR
5
, with only GFR
1
180 giv-
ing a clinically acceptable margin of error. Furthermore, when collect-
ing only 1 sample any analytical error will influence the accuracy of
the clearance measurement. However, the advantage of reducing
physical discomfort and stress for the dog, as well as costs and time,
substantially balances the risk of errors in GFR measures. In addition,
a gray-zone approach with an interval of uncertainty for overlapping
values can reduce the potential for error and thus limit wrong clinical
decisions. Indeed, this could lead to a decision to use further diagnos-
tic tools in a smaller group with inconclusive results and, at the same
time, offer diagnostic certainty outside the gray zone.
The assessment of GFR from iohexol plasma clearance in dogs
still suffers a lack in standardization. Differences in the marker
used, the marker concentration assays, sampling times, pharmaco-
kinetic models, and mathematical modeling of the data used for
calculating GFR can all lead to wide variability in the GFR reported
for healthy and diseased dogs.
7,24,37
We investigated 2 controver-
sial issues hindering the harmonization of GFR measurement in
dogs between laboratories: which of the 2 iohexol isomers
detected by HPLC-UV has to be used for plasma clearance calcula-
tion and which measure of body size has to be used for GFR
normalization.
Discrepancies have been reported by calculating GFR based on
the plasma clearance of endo- and exo-iohexol separately
6,8,19,22,23
and the use of the total iohexol.
4
Here, we found that in plasma
samples frozen for a long time, a significant proportion of the endo-
iohexol was shifted to the exo-iohexol. This is in agreement with
early reports that the isomers are interconvertible and that rota-
tional conversion is temperature-
38
and storage-dependent.
39
The
isomer shift modified single peak areas but did not influence their
sum, in agreement with reports that total iohexol is very stable in
plasma.
13,40,41
For GFR measurement in veterinary practice, injec-
tion of the tracer and sample analysis are often separate in space
and time, so stability is of primary importance. Our results suggest
the routine use of the sum of the absorbance peaks of the 2 isomers
for calculating iohexol clearance in order to avoid preanalytical
errors, especially if the samples need to be frozen or sent by mail to
the reference laboratory.
ThemostcommonlyusedmethodtonormalizeGFRindogsis
indexation to BW, but some authors prefer to normalize the mea-
surements to BSA,
9,10,16
and differences have been reported
between the levels of renal function standardized to BW or BSA.
4
In
the present study, we observed that the correlation between iohexol
plasma clearance and dog body size was lost only when GFR was
scaled to BW. This supports the recommendation that indexation to
BSA should be abandoned and that the formulae used to estimate
BSA in dogs is of questionable accuracy.
42,43
Our analyses were
done on dogs with a wide range and normal BW distribution, but the
group was only small, the majority weighing 25-27 kg. This might be
a bias when drawing any conclusion that BW is a better method for
clearance indexation in dogs. Whether our findings are valid for a
general population of dogs with and without CKD needs further
study.
In conclusion, we propose a panel of accurate, hands-on, flexible,
simplified procedures for estimating GFR in dogs as a practical tool for
CKD diagnosis in daily clinical practice. We also recommend the gray
zone concept of uncertainty in CKD diagnosis, as it can be especially
useful when it is more important to suspect reduced renal function as
early as possible than to know the exact GFR.
ACKNOWLEDGMENTS
The authors acknowledge Mr. Valerio Lorenzo Muci for valuable assis-
tance in this work.
GFR
5
CKD- CKD+
0.0
1.0
2.0
3.0
4.0
GFR (mL/min/kg)
GFR
3
CKD- CKD+
0.0
1.0
2.0
3.0
4.0
GFR (mL/min/kg)
GFR
2
60-180
CKD- CKD+
0.0
1.0
2.0
3.0
4.0
GFR (mL/min/kg)
GFR
2
90-180
CKD- CKD+
0.0
1.0
2.0
3.0
4.0
GFR (mL/min/kg)
GFR
1
180
CKD- CKD+
0.0
1.0
2.0
3.0
4.0
GFR (mL/min/kg)
FIGURE 8 Scatter plot of the glomerular filtration rate (GFR) in
CKD+ (white dots) and CKD−(black dots) using different GFR
measurement protocols. Median and interquartile ranges are shown
as horizontal lines. The gray zone illustrates the range of CKD
diagnostic uncertainty. CKD, chronic kidney disease
2114 POCAR ET AL.
CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.
OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.
INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE
(IACUC) OR OTHER APPROVAL DECLARATION
Approval from Animal Care Committee of the University of Milan,
opinion_n.107_2016.
HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.
ORCID
Paola Pocar https://orcid.org/0000-0002-1362-001X
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How to cite this article: Pocar P, Scarpa P, Berrini A,
Cagnardi P, Rizzi R, Borromeo V. Diagnostic potential of
simplified methods for measuring glomerular filtration rate to
detect chronic kidney disease in dogs. J Vet Intern Med. 2019;
33:2105–2116. https://doi.org/10.1111/jvim.15573
2116 POCAR ET AL.
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