Thanaboonnipat et al.

BMC Veterinary Research (2019) 15:54

https://doi.org/10.1186/s12917-019-1801-4

RESEARCH ARTICLE Open Access

Renal shear wave elastography and urinary

procollagen type III amino-terminal
propeptide (uPIIINP) in feline chronic
kidney disease
Chutimon Thanaboonnipat1,2, Saikaew Sutayatram1, Chollada Buranakarl1 and Nan Choisunirachon2*

Abstract
Background: Chronic kidney disease (CKD) is one of the most common diseases occurring in cats. It is characterized
by renal fibrosis, which is strongly correlated with impairment of renal function. Since renal biopsy is not performed
routinely in clinical practice, the non-invasive method of ultrasonographic shear-wave elastography (SWE) was used to
determine renal parenchymal stiffness. Currently, urinary procollagen type III amino-terminal propeptide (uPIIINP) is a
renal fibrosis biomarker in humans. Moreover, PIIINP is increasingly applied for identification of fibrosis in various organs
in animals.
Results: The Young’s modulus (E) value on SWE, uPIIINP, and renal function were evaluated in 23 CKD cats and 25
healthy cats (HC). The renal cortical E values were significantly higher than those of the renal medulla in both groups
(P < 0.001). The E values of the renal cortex and medulla were significantly higher in CKD cats than in HC (P < 0.001 and
P < 0.01, respectively). The E values, especially of the cortex, showed a significant positive correlation with concentrations
of plasma creatinine (P < 0.001), blood urea nitrogen (P < 0.05), while they had a negative correlation with urine specific
gravity (P < 0.001) and urine osmolality per plasma osmolality ratio (P < 0.01). The uPIIINP to creatinine ratios
(uPIIINP/Cr) were significantly higher in CKD cats than in HC (P < 0.01) and were highly correlated with renal
cortical E values (P < 0.001).
Conclusions: SWE might be an additively useful and non-invasive diagnostic imaging tool to evaluate renal
parenchymal stiffness, which correlates with renal functional impairment in CKD cats. Moreover, the uPIIINP/Cr
might be a promissing biomarker for adjunctive assessing the renal fibrosis in feline CKD.
Keywords: Cat, Chronic kidney disease (CKD), Urinary procollagen type III amino-terminal propeptide (uPIIINP),
Renal fibrosis, Shear wave elastography (SWE)

Background biopsy is the gold standard for diagnosing renal fibrosis

Chronic kidney disease (CKD) is a common disease in cats [8]. However, it requires anesthesia and can cause several
and has been increasingly diagnosed annually, particularly complications. The most common complication of renal
in aging cats [1–5]. Renal fibrosis is the hallmark of ad- biopsy is hemorrhage. Therefore, it is not performed rou-
vanced CKD and strongly correlates with renal function tinely in clinical practice particularly in high-risk patients
impairment in humans and cats [6, 7]. Renal fibrosis such as CKD cats [9].
causes an irreversible loss of normal tissue, diminished Cats with CKD normally display non-specific signs
renal function, and end-stage renal disease (ESRD). Renal [10, 11], and routine laboratory tests, such as blood cre-
atinine concentration, are not sensitive enough for early
* Correspondence: [email protected]
2
CKD detection [12]. Abdominal ultrasonography can
Department of Veterinary Surgery, Faculty of Veterinary Science,
Chulalongkorn University, 39 Henri-Dunant Road, Wangmai, Pathumwan,
provide information on the renal parenchyma that is im-
Bangkok 10330, Thailand portant for identifying the underlying causes of CKD.
Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
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Thanaboonnipat et al. BMC Veterinary Research (2019) 15:54 Page 2 of 10

However, typical ultrasonographic findings, such as Committee (CU-ACUC), Faculty of Veterinary Science,
small and irregular kidneys or increased echogenicity of Chulalongkorn University (Protocol number: 1731055).
the cortex and/or medulla [13–16], are not specific for All client-owned cats, including healthy cats (HC) and
CKD, and are sometimes found in healthy cats [17]. CKD cats that presented to the Small Animal Teaching
Several methods are currently used in human medicine Hospital, Faculty of Veterinary Science, Chulalongkorn
for the determination of renal fibrosis. One of the renal University from September to December 2017, were
fibrosis biomarkers is urinary procollagen type III considered for inclusion. The cats were considered to be
amino-terminal propeptide (uPIIINP) [8, 18]. Previous HC if the history, physical examination, routine blood
studies in people have reported that high urine PIIINP to examinations, including complete blood count (CBC)
creatinine ratio (uPIIINP/Cr) was highly associated with and plasma biochemistry, USG, urinalysis, UPC ratio,
the severity of renal fibrosis and CKD progression [8, 19, abdominal radiographs, and abdominal ultrasound were
20]. Moreover, in animals, PIIINP was also used as a fibro- normal. All cats tested negative for Feline Immunodefi-
sis marker for assessing cardiac remodeling [21], idiopathic ciency Virus (FIV) and Feline Leukemia Virus (FeLV) in-
pulmonary fibrosis [22], and liver fibrosis in dogs [23]. fection using ELISA Test Kits (WITNESS® FeLV-FIV,
In addition, renal ultrasonographic elastography (USE) Zoetis, New Jersey, USA). The CKD cats included in this
is an imaging technique that has been increasingly applied study were defined based on a history of either structural
in human medicine [24, 25]. USE can evaluate tissue elas- or functional abnormalities of the kidneys for more than
ticity by differentiating between normal tissue and stiff tis- 3 months (stable CKD patients) and were categorized
sue, which cannot be done using hyperechogenic images into IRIS stage 2, 3 and 4 CKD according to the Inter-
from B-mode ultrasonography [26]. USE examines the tis- national Renal Interest Society (IRIS) [35] staging sys-
sue hardness [27, 28], using tissue elasticity evaluation that tem, in which the plasma creatinine concentrations
is inversely proportional to stiffness [29]. As previously de- equal to or higher than 1.6 mg/dl. Additionally, all CKD
scribed, fibrous tissue infiltration in the renal parenchyma cats underwent the same diagnostic procedures as the
results in decreased elasticity [30]. The two main tech- HC and had negative results for FIV and FeLV infection
niques of USE available are strain elastography (SE) and from ELISA Test Kits (WITNESS® FeLV-FIV, Zoetis,
shear wave elastography (SWE) [26]. In human medicine, New Jersey, USA). Cats with ascites, infectious diseases,
SWE has been widely performed to assess the renal paren- lymphoma, congenital kidney diseases, such as polycystic
chymal stiffness through the Young’s modulus (E) in CKD kidney disease, hydronephrosis, generalized peripheral
patients [25, 31]. Several studies have reported that human edema, or nephroliths were excluded from this study.
patients with CKD had renal E values significantly higher A total of 3 ml of blood was collected from each cat
than those of healthy individuals [25, 31]. Therefore, renal and placed in an EDTA tube for complete blood count
USE shows promise as a diagnostic imaging method to de- (CBC) and a heparinized tube for measurement of cre-
tect the early stage of CKD [28, 32, 33]. atinine concentration, BUN, alanine aminotransferase
In veterinary medicine, especially in cats, the use of (ALT), alkaline phosphatase (ALP), total protein, albu-
uPIIINP has not been reported, and reports on the use min, osmolality, and electrolytes (Na+, K−, Cl− and Mg2
+
of USE are limited and only include the use of SE on ). Urine was collected by voiding or catheterization for
normal kidneys [34]. Therefore, the purposes of this measuring the concentrations of creatinine, protein,
study were, first, to compare the renal tissue stiffness ob- electrolytes (Na+, K−, Cl− and Mg2+), PIIINP, and osmo-
served through E values between healthy cats and CKD lality. Indirect blood pressure in all cats was measured
cats using SWE; second to evaluate the relationships be- three times at the proximal hind leg or distal forelimb
tween the renal tissue stiffness and functional renal pa- and the results were averaged. All ultrasonographic pro-
rameters, including plasma creatinine concentration, cedures were performed within a visit day.
blood urea nitrogen (BUN), urine specific gravity (USG),
urine protein creatinine (UPC) ratio, urine osmolality to Analytical procedures
plasma osmolality (Uosm/Posm) ratio, and fractional ex- Concentrations of creatinine, BUN, ALT, ALP, total pro-
cretion of sodium, potassium, chloride, and magnesium tein, and albumin were determined within an hour of
(FENa, FEK, FECl, and FEMg) in both groups; and third to collection by automated analyzer (The IL ILab 650
determine the relationship between uPIIINP/Cr levels Chemistry Analyzer, Diamond Diagnostic, MA, USA).
and blood pressure, UPC ratio or renal E values. Frozen plasma and frozen urine samples (− 20 °C) were
thawed to measure the osmolality and electrolytes con-
Methods centration within a few weeks of collection. The plasma
General materials and urine osmolality were measured using automatic
This clinical, cross-sectional study was approved by the cryoscopic osmometer (OSMOMAT® 030, Gonotec
Chulalongkorn University Animal Care and Use GmbH, Berlin, Germany), the plasma concentrations of
Thanaboonnipat et al. BMC Veterinary Research (2019) 15:54 Page 3 of 10

electrolytes were analyzed using automated analyzer The E value in kilopascal (kPa) unit was selected for
(ARCHITECT c16000 clinical chemistry analyzer, Abbott representation of the tissue stiffness [37]. A stiff material
Laboratories, IL, USA) and the urine concentrations of has a higher E value than a soft material [37, 38]. Three
electrolytes were analyzed using automated analyzer separate elastograms were selected, and E values were
(COBAS INTEGRA® 400 plus, Roche Diagnostics, Rotk- measured at the superficial, mid renal parenchymal re-
reuz, Switzerland). USG was determined within an hour gion using a circular electrical caliper which circular size
of collection using a refractometer (Master Refractom- varied according to the thickness of the renal cortical
eter; ATAGO®, Tokyo, Japan). Measurement of blood and medullary parenchyma in both HC and CKD cats
pressure was performed using a Doppler blood pressure (Fig. 1), and the measurements from each elastogram
instrument (Vet-Dop 2™, Vmed Technology, WA, USA). were averaged.
The microscopic observation of the urine sediment was
performed prior to the measurement of the UPC to ex- Statistical analysis
clude the urinary tract infection. The UPC ratio was per- Statistical analyses were performed by GraphPad Prism
formed by measuring urine protein concentrations after 7 software (GraphPad Software; CA; USA). Results were
precipitation with 3% sulphosalicylic acid [36]. Fractional expressed as mean ± standard deviation (SD). Normality
excretions (FENa, FEK, FECl, and FEMg) were calculated distributions were tested with the Shapiro–Wilk test.
using the formula as previously described [36]. Urinary The average E value results between left and right kid-
PIIINP concentrations were determined in frozen urine neys in each group were analyzed by paired t-test. The
samples (− 80 °C) within 2 months of collection using a average E values and uPIIINP/Cr levels were compared
sandwich enzyme-linked immunosorbent assay (ELISA) between groups by the unpaired t-test or Mann Whitney
kit (Cat Procollagen Type III N-Terminal Propeptide test. Pearson’s correlation coefficient and Spearman’s
(PIIINP) ELISA Kit, Cat. No. MBS060292, MyBio- correlation coefficient were used to investigate the corre-
Source®, CA, USA), following the manufacturer’s instruc- lations between parameters. Statistical significance was
tions. Urine samples were assayed in duplicate, with an considered if the P-value was less than 0.05.
intra-assay and inter-assay coefficient of variation less
than 15%. The uPIIINP/Cr levels were calculated and Results
expressed as ng/mgCr. A total of 25 HC and 23 CKD cats were included in this
study. The mean ages of the HC and CKD cats were 4.5
± 3.4 years (median 3 years; range 8 months–10 years)
SWE procedure and 9.7 ± 5.1 years (median 9 years; range 3–21 years),
The ultrasonographic examinations were performed respectively. There were 11 females (4 intact and 7 neu-
without sedation. The cats were manually restrained and tered) and 14 males (6 intact and 8 neutered) in the HC
positioned in right and left lateral recumbency for each group, and there were 15 females (10 intact and 5 neu-
ipsilateral site of kidneys. Routine preparation for ultra- tered) and 8 males (2 intact and 6 neutered) in the CKD
sonographic examination, including hair clipping and group. The mean bodyweight of HC was 4.1 ± 0.9 kg
acoustic gel coupling, was performed. All cats were (range 2.9–5.9 kg), while the mean body weight of CKD
screened with B-mode abdominal ultrasound, and the cats was 3.7 ± 0.9 kg (range; 2.6–5.9). The body condi-
sagittal, dorsal, and transverse planes of the kidneys were tion scores (BCS) of both groups were comparable and
recorded using a 9 MHz-bandwidth of a linear trans- ranged from a score of 2–4 out of 5 in each group, with
ducer (Resona-7, Mindray Medical International, Shen- a mean of 3.2 ± 0.6 and 3.0 ± 0.4 for HC and CKD cats,
zhen, China). SWE was then performed on the sagittal respectively. The HC consisted of Domestic shorthair
plane of each kidney. The elastograms were displayed (18), Persian (5), and American shorthair (2), and the
using a dual-screen, consisting of a B-mode image and CKD cats were Domestic shorthair (22) and Persian (1).
an SWE color quantitative elastogram overlaid on The CKD cats were belonging to IRIS stage 2 (82.6%),
B-mode. The fixed region of interest (ROI) was adjusted stage 3 (13.1%) and stage 4 (4.3%).
as wide as possible to cover both the renal cortex and Ultrasonographically, the kidneys in HC had normal
renal medulla while attempting to control the high reli- shape, contour, echogenicity, and echotexture, with a
ability (RLB) map during the procedure. The RLB map mean length of 3.6 ± 0.4 and 3.5 ± 0.4 cm for the right
indicates the quality of shear wave image in a number and left kidneys, respectively. In contrast, CKD cats
percentage. The elasticity results measured from shear had irregular renal outlines, increased echogenicity of
wave images with higher RLB percentages are more reli- the cortex and/or medulla, and decreased corticome-
able. The elastogram color scale expressed a red color to dullary demarcation. The CKD cats also had signifi-
represent the hardest tissues, whereas a blue color repre- cantly shorter renal length (mean length of 3.1 ± 0.5
sented the softest tissues. and 3.1 ± 0.6 cm for the right and left kidneys,
Thanaboonnipat et al. BMC Veterinary Research (2019) 15:54 Page 4 of 10

Fig. 1 B-mode ultrasound images; a, c and color quantitative elastogram overlaid on the ROI on the B-mode; b, d between a healthy cat (HC); a,
b comparing to a chronic kidney disease (CKD) cat; c, d. Regions of interest (ROIs) were drawn over the mid renal cortex and medulla to measure
the Young’s modulus (E) value

respectively) than the HC (Right kidney; P < 0.01 and Regarding gender, the average renal E values of male cats
Left kidney; P < 0.001). and female cats were not significantly different. How-
The average blood pressure of HC group was signifi- ever, average renal cortical E values were significantly
cantly lower than that of CKD cats (129.2 ± 17.3 mmHg higher than those of the renal medulla in both groups
and 150.9 ± 26.4 mmHg, respectively; P < 0.01). Urine (P < 0.001). Moreover, the average E values of the renal
collection was achieved in 39 cats, which were 19 HC, cortex and medulla of CKD cats were significantly
and 20 CKD cats. The functional renal parameters of higher than those of HC (P < 0.001 and P < 0.01, re-
both groups, including plasma creatinine concentration, spectively) (Fig. 2).
BUN, USG, UPC ratio, Uosm/Posm ratio, FENa, FEK, The correlations between average renal E values and
FECl, and FEMg, are summarized in Table 1. Plasma cre- plasma creatinine concentrations or BUN in the 48 cats
atinine, BUN concentration and UPC ratio of CKD cats from both groups, and the correlations between the
were significantly higher than those of HC (P < 0.001), average renal E value and USG, UPC ratio, Uosm/Posm
while USG and Uosm/Posm ratio of CKD cats were sig- ratio, FENa, FEK, FECl, or FEMg in 39 cats, from both the
nificantly lower than those of HC (P < 0.001). All FE of HC and CKD groups, were evaluated (Table 3). The E
electrolytes, except FENa, were significantly higher in values of the cortex showed a significant positive correl-
CKD cats than in HC (P < 0.001). ation with concentrations of plasma creatinine (P < 0.001),
The renal elasticity on the SWE of the 25 HC and 23 BUN (P < 0.05), and FECl (P < 0.01), while it showed a
CKD cats were reported as average E values calculated negative correlation with USG (P < 0.001) and Uosm/
from the right and left kidneys. The average E values of Posm (P < 0.01) (Fig. 3).
the renal cortex and medulla were not significantly dif- The average uPIIINP/Cr values of 17 HC and 20 CKD
ferent between the right and left kidneys in each group cats were compared, and the results showed that CKD
(Table 2). Age, bodyweight, and BCS did not affect the cats had average uPIIINP/Cr values significantly higher
average E values of the cortex or medulla in HC. than those of HC (mean uPIIINP/Cr of 34.9 ± 29.4 and
Thanaboonnipat et al. BMC Veterinary Research (2019) 15:54 Page 5 of 10

Table 1 Functional renal parameters include concentrations of plasma creatinine and BUN, USG, UPC ratio, Uosm/Posm ratio and
FENa, FEK, FECl, FEMg of the healthy cats (HC) and chronic kidney disease (CKD) cats
Parameters No. of s samples Healthy cats CKD cats Reference intervals
***
Plasma creatinine (mg/dL) 48 1.36 ± 0.19 2.59 ± 0.99 0.6–1.6
BUN (mg/dL) 48 22.39 ± 4.62 39.74 ± 22.79*** 14–36
***
USG 39 1.055 ± 0.01 1.034 ± 0.015 1.035–1.060
UPC ratio 9 0.09 ± 0.1 0.96 ± 1.71*** < 0.2a
Uosm/Posm ratio 39 5.95 ± 1.56 2.95 ± 2.12*** NA
FENa (%) 39 0.58 ± 0.29 2.57 ± 3.02 < 1
***
FEK (%) 39 18.39 ± 6.41 44.69 ± 27.41 < 20
FECl (%) 39 1.08 ± 0.47 4.79 ± 4.56*** < 1
***
FEMg (%) 39 2.41 ± 1.57 9.11 ± 7.86 < 5.4
a
Reference of UPC ratio in cats = non-proteinuric (< 0.2), borderline (0.2–0.4), proteinuric (> 0.4)
BUN blood urea nitrogen, USG urine specific gravity, UPC ratio urine protein creatinine ratio, Uosm/Posm ratio urine osmolality per plasma osmolality ratio, FENa
fractional excretion of sodium, FEK fractional excretion of potassium, FECl fractional excretion of chloride, FEMg fractional excretion of magnesium
NA denotes no available data
Data are presented as mean ± SD
***
Statistically difference between groups was made using Unpaired t- test, P < 0.001

8.1 ± 5.2 ng/mgCr for CKD cats and HC, respectively; P SWE was performed in this study because it is the
< 0.01) (Fig. 4). No significant correlations were pre- newest USE technique [44]; it also provides better infor-
sented between uPIIINP/Cr and blood pressure or UPC mation on elasticity than SE, in which the result is
ratio. A significant positive correlation (P < 0.001) was highly dependent upon operator experience [45]. Other
found between uPIIINP/Cr levels and average renal cor- imaging techniques have also been used to facilitate an
tical E values (Fig. 5), while uPIIINP/Cr levels were not early CKD diagnosis in cats, such as: contrast-enhanced
significantly correlated with average renal medullary E ultrasound examination in domestic and purebred cats
values. [46], and renal resistive index (RRI) in domestic cats
[47]. However, methods such as RRI are difficult to
evaluate, especially in CKD cats with renal vascular im-
Discussion pairment and decreased renal blood flow [47]. Peng and
USE can differentiate normal tissue from pathological colleagues [25] reported that human patients with CKD
tissue with an altered elasticity and increased stiffness, had stiffer kidneys than healthy people, when using
such as a fibrotic process [25, 39]. In veterinary medi- SWE. Therefore, SWE may be more useful for the inves-
cine, the use of elastography has been described in a tigation of renal parenchymal stiffness than conventional
small number of studies on normal kidneys of various B-mode ultrasound, especially in CKD cats.
dog breeds [29] including beagles [40] and various cat
breeds [34]. It has also been used to study canine mam-
mary tumors [41, 42], and canine malignant lymph
nodes [43]; however, it has not been used in renal
disease.

Table 2 The Young’s modulus (E) values (kPa) of renal

parenchyma between healthy cats group and CKD cats group
Group E value of LK E value of RK
Cortex Medulla Cortex Medulla
HC 38.40 ± 7.12 30.16 ± 5.97 40.34 ± 10.42 32.73 ± 7.04
(22.91–55.58) (19.43–40.13) (23.70–53.74) (18.27–49.30)
CKD 51.89 ± 11.25 39.16 ± 13.07 51.97 ± 12.65 36.60 ± 10.06
(22.55–74.42) (20.19–82.13) (23.38–75.60) (15.38–66.51)
E value the Young’s modulus values in kilopascal unit, LK left kidney, RK right Fig. 2 Average renal E values of cortex and medulla (mean ± SD)
kidney, HC healthy cats group, CKD chronic kidney disease cats group between the healthy cats (HC) and chronic kidney disease (CKD). ***
Data are expressed as mean ± SD and range Unpaired t-test, P < 0.001; ** Mann-Whitney test, P < 0.01. E = Young’s
The average E values between left and right kidneys in each group were modulus values
analyzed by Paired t-test
Thanaboonnipat et al. BMC Veterinary Research (2019) 15:54 Page 6 of 10

Table 3 Correlations between the average renal Young’s As previously described, the renal length in cats may
modulus (E) values and plasma creatinine concentration, BUN, vary with gonadal status [48]. However, in this study,
USG, UPC ratio, Uosm/Posm ratio and FENa, FEK, FECl, FEMg in the both groups contained similar numbers of intact and
healthy cats (HC) and chronic kidney disease (CKD) cats neutered cats. Thus, the shorter average renal length in
Parameters No. of Average E values Average E values CKD cats compared with that in HC cats was mainly
samples of cortex of medulla
due to disease progression, as decreased renal size is a
r r common finding in advanced CKD [15].
***
Plasma creatinine 48 0.509 0.329* In contrast with the results from previous studies in
*
BUN 48 0.361 0.188 canine, the E values did not vary between the left and
USG 39 −0.567 ***
−0.255 right kidneys in either group of cats. This might be be-
UPC ratio 39 0.233 −0.045 cause the location of the feline kidney, particularly in
*** right kidney, interferes less with the SWE procedure
Uosm/Posm ratio 39 −0.582 −0.306
compared with the location of right kidney in dogs,
FENa 39 0.256 0.089
which is more cranial and nearer to the rib cage [40, 49].
FEK 39 0.250 −0.219 Therefore, respiratory motion was observed to be more
FECl 39 0.506** 0.478** problematic when performing the SWE of the right kid-
FEMg 39 0.293 0.079 ney in comparison to the left kidney in dogs [29].
E value the Young’s modulus values, BUN blood urea nitrogen, USG urine In agreement with the findings of previous studies in
specific gravity, UPC ratio urine protein creatinine ratio, Uosm/Posm ratio urine dogs and humans [29, 50], our results show that age, gen-
osmolality plasma osmolality ratio, FENa fractional excretion of sodium, FEK
fractional excretion of potassium, FECl fractional excretion of chloride, FEMg
der, bodyweight, and BCS did not affect the renal E values
fractional excretion of magnesium in HC cats. However, in this study, the small number of
Correlations between parameters were made using Spearman correlation,*P < animals is an important limitation in evaluating the
0.05; **P < 0.01; ***P < 0.001
influence of demographic variables on elastography. In
addition, the ages between groups were unmatched. The
HC group was younger than the CKD group, because

Fig. 3 Correlations between renal cortical E values and other renal parameters using Spearman correlation; a average renal cortical E values and
plasma creatinine; b average renal cortical E values and USG; c average renal cortical E values and Uosm/Posm; d average renal cortical E values
and FECl. E = Young’s modulus values; USG = Urine specific gravity; Uosm/Posm = Urine osmolality per plasma osmolality; FECl = Fractional
excretion of chloride
Thanaboonnipat et al. BMC Veterinary Research (2019) 15:54 Page 7 of 10

blood supply to the kidneys. Tissue elasticity is highly in-

fluenced by the degree of vascular pressure, and a greater
proportion of the cardiac blood flow is distributed to the
renal cortex than to the renal medulla [27].
Both the renal cortical and medullary E values of CKD
cats were significantly higher than those of HC cats,
which demonstrated that CKD cats had increased kidney
stiffness. These results correspond with the studies by
Guo et al. [28], Hu et al. [32], Samir et al. [33], and Peng
et al. [25] in humans with CKD. However, the results of
this study showed that there was an overlap between the
average renal E values of HC cats and CKD cats. This
might limit the use of USE for the definitive diagnosis of
CKD. Rather, USE should be used as an adjunctive tool
Fig. 4 Box and whisker plot illustrating uPIIINP/Cr between the healthy
for more accurate detection and more informative moni-
cats (HC) and chronic kidney disease (CKD). * Statistically difference
between groups was made using Mann-Whitney test, P < 0.01. uPIIINP/ toring of feline CKD. Furthermore, USE might assist in
Cr = Urine Procollagen Type III N-Terminal Propeptide to creatinine ratio the diagnosis and treatment planning of renal disease in
subclinical cases.
It is commonly known that renal tubulointerstitial fi-
CKD has high incidence in senior cats. Therefore, the abil- brosis is the final common pathway of kidney disease
ity to obtain a HC group with a comparable age to that of [51], and is highly correlated with impairment of renal
the CKD group is challenging. function in both humans [52, 53] and cats [54, 55].
The elasticity of the renal cortex was significantly less Renal tubular functions including the absorption and ex-
than that of the renal medulla in both groups, which cor- cretion of water and electrolytes, were evaluated by
responds to SWE studies conducted in humans [25, 27] measuring the Uosm/Posm ratio, FENa, FEK, FECl, and
and pigs [45]. In contrast, the opposite results were re- FEMg in this study.
ported in SE studies of healthy dogs [40] and healthy cats The results revealed that renal cortical E values had
[34]. This disagreement among studies might result from significant positive correlations with plasma creatinine
the differences in ultrasound modalities and picture acqui- concentrations, BUN, and FECl, while significant nega-
sition techniques. Further studies that compare between tive correlations existed with USG and Uosm/Posm ra-
techniques would provide more clinical information of tio. These results correspond with the study by Lin et al.
renal elasticity. From our findings, the lower elasticity of [24] in humans, in which renal elasticity was associated
the renal cortex compared with the renal medulla could with a deterioration of renal function in patients with
be explained by the difference in the normal physiological CKD. In this study, CKD cats exhibited a urinary con-
centrating defect leading to lower urine osmolality and
Uosm/Posm ratio than in HC cats. Moreover, fractional
excretion of electrolytes (FEe) has been reported to be a
highly sensitive parameter for the evaluation of tubular
impairment in dogs with advanced CKD [36]. Addition-
ally, it has been demonstrated that FEMg may be a useful
marker of the severity of tubular cell damage, as it has
been positively correlated with the stage of tubulointer-
stitial fibrosis in humans [56].
Additionally, our findings indicate that the renal cor-
tical E value was predominantly correlated with renal
functions compared with that of the renal medullary E
value. The reason for this could be that the renal cortex
contains the glomeruli, which are responsible for plasma
filtration [57], as well as the proximal tubules, which are
responsible for the reabsorption and secretion of sub-
Fig. 5 Correlation between average renal cortical E values and uPIIINP/ stances, particularly of electrolytes [58].
Cr of 37 cats. Correlation was made using Pearson’s correlation However, the urine osmolality and FEe vary depending
coefficient. E = Young’s modulus values; uPIIINP/Cr = Urine Procollagen
on several exogenous and endogenous factors especially
Type III N-Terminal Propeptide to creatinine
diet and hydration status [59]. In this study, we attempted
Thanaboonnipat et al. BMC Veterinary Research (2019) 15:54 Page 8 of 10

to avoid the influence of hydration status through complete In veterinary medicine, information concerning renal
history taking and physical examination, and only included function parameters and renal elasticity in cats has not
normally hydrated cats in our study. However, the effect of been reported. Moreover, the relationship between the
food on these parameters could not be controlled in this uPIIINP/Cr level and renal elasticity in CKD cats has not
study, as the use of a prescription diet is a clinical recom- been determined. This is the first report relating the renal
mendation in the management of CKD. Therefore, this elasticity, as determined with SWE, to the uPIIINP/Cr in
might be an additional limitation of our study. CKD cats. This information would be useful for clinical
The uPIIINP/Cr ratio, which was strongly correlated practitioners and would be of interest to researchers in
with the progression of interstitial fibrosis in kidney bi- further study.
opsies from human patients [8, 18, 19], was significantly The major limitation of this study is that we did not per-
higher in CKD cats than in HC cats. This corresponds form a renal histopathological examination for compari-
to the results of human studies [8, 18, 19], and the re- son with the renal elasticity determined by USE and the
sults of canine studies that used PIIINP as a marker of uPIIINP/Cr results, due to the invasive nature of the bi-
fibrosis in cardiac remodeling [21], idiopathic pulmonary opsy. Therefore, further studies should be considered. The
fibrosis [22], and liver fibrosis [23]. Moreover, no signifi- second limitation is that the number of healthy cats in-
cant correlation was found between the uPIIINP/Cr and cluded in the study was not sufficient to evaluate the influ-
blood pressure or UPC ratio. This also corresponds to ence of demographic variables on SWE. Additionally, the
the results of human research, in which uPIIINP was HC group and CKD group were not age-matched. Further
not dependent on the degree of proteinuria [19]. study into the effect of age and BCS on the renal elasticity
Urinary transforming growth factor beta 1 (TGF-β1) is would provide more reliable information. The third limita-
considered the most popular mediator for detecting the tion is that other uncontrollable factors of client-owned
severity of renal fibrosis in cats [60]. However, the cats, such as different types of commercial foods, might
current gold standard method for identifying fibrosis is influence the Uosm/Posm ratio and FEe results in this
still a kidney biopsy [8, 61]. Although, it has been re- study. A further limitation is that we could not collect
ported that urinary TGF-β1 concentration is increased urine samples from all cats.
in CKD cats [62, 63] and is correlated with histopatho-
logical evidence of interstitial fibrosis in cats [60], it has Conclusions
also been reported that urinary TGF-β1 shows no sig- USE can be used an additive diagnostic imaging method
nificant association with worsening renal function in for the evaluation and monitoring of feline CKD. Plasma
both humans [61] and cats [60]. While, uPIIINP has creatinine, BUN, and FECl had a positive correlation with
been evaluated in human patients at various stages of renal elasticity, while USG and Uosm/Posm ratio revealed
CKD, the uPIIINP/Cr has been correlated with the de- a negative correlation with renal elasticity. The renal cor-
gree of renal function impairment, as well as with the se- tex in both groups had lower elasticity than the renal me-
verity of renal fibrosis [8, 18–20]. In addition, uPIIINP dulla, and the kidneys of CKD cats were stiffer than those
levels correlated with urinary TGF-β1 in human studies of HC in this study. Moreover, the renal cortical E values
[19]. Therefore, uPIIINP has been considered as one of were significantly correlated to the uPIIINP/Cr levels. The
the reliable markers of renal fibrosis in humans [8, 19]. results suggest that renal SWE might be an easy-to-use
Furthermore, as PIIINP has a low molecular weight (42 diagnostic tool, which may be applied as adjunct for the
kDa), it is filtered by the glomeruli and reabsorbed in detection and monitor of feline CKD. Furthermore, the
the proximal tubules [8, 18, 19]. Thus, an elevation in uPIIINP/Cr levels are higher in CKD cats. Therefore, the
uPIIIINP could indicate reduced tubular reabsorption uPIIINP/Cr might also be a promising biomarker for ad-
capacity due to the progression of renal fibrosis. More- junctive assessing the renal fibrosis in CKD cats. However,
over, it has previously been shown in human studies [19] further studies are needed to elucidate the clinical value of
and in our study that the level of uPIIINP is not related USE in terms of evaluation and monitoring of feline CKD
to the degree of proteinuria. It might be of use as an ad- and to construct a correlation between uPIIINP/Cr and
junct biomarker for the detection of renal fibrosis in degree of renal fibrosis from histopathology in cats.
cats; however, the value of uPIIINP at each stage of renal
fibrosis and the concomitant histopathology in cats is Abbreviations
currently still unknown. BCS: Body condition score; BUN: Blood urea nitrogen; CBC: Complete blood
count; CKD: Chronic kidney disease; CU-ACUC: Chulalongkorn University
In this study, a statistically positive and moderate cor- Animal Care and Use Committee; E: The Young’s modulus (E) value Standard
relation was found between the uPIIINP/Cr level and deviation; ECM: Extracellular matrix; ESRD: End-stage renal disease;
the average renal cortical E values, suggesting that the FEe: Fractional excretion of electrolyte; FENa, FEK, FECl, and FEMg: Fractional
excretion of sodium, potassium, chloride, and magnesium; HC: Healthy cats;
renal cortical E value might be one of the indicators to kPa: kilopascal; mmHg: Millimeter of mercury; RLB: Reliability; ROI: Region of
assist in the evaluation of renal fibrosis in CKD cats. interest; SD: Standard deviation; SE: Strain elastography; SWE: Shear-wave
Thanaboonnipat et al. BMC Veterinary Research (2019) 15:54 Page 9 of 10

elastography; TGF-β1: Transforming growth factor beta 1; Uosm/Posm: Urine 7. Lee SY, Kim SI, Choi ME. Therapeutic targets for treating fibrotic kidney
osmolality to plasma osmolality ratio; UPC: Urine protein creatinine; diseases. Transl Res. 2015;165:512–30.
uPIIINP: Urinary procollagen type III amino-terminal propeptide; uPIIINP/ 8. El Ghoul B, Squalli T, Servais A, Elie C, Meas-Yedid V, Trivint C,
Cr: Urine PIIINP to creatinine ratio; USE: Ultrasonographic elastography; Vanmassenhove J, Grünfeld JP, Olivo-Marin JC, Thervet E, Noël LH, Prié D,
USG: Urine specific gravity Fakhouri F. Urinary procollagen III aminoterminal propeptide (PIIINP): a
fibrotest for the nephrologist. Clin J Am Soc Nephrol. 2010;5:205–10.
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10. DiBartola SP, Rutgers HC, Zack PM, Tarr MJ. Clinicopathologic findings
Funding associated with chronic renal disease in cats: 74 cases (1973-1984). J Am Vet
This study was granted from the 100th Anniversary Chulalongkorn University Med Assoc. 1987;190(9):1196–202.
for Doctoral Scholarship and the 90th Anniversary of Chulalongkorn 11. King JN, Tasker S, Gunn-Moore DA, Strehlau G. Prognostic factors in cats
University Fund (Ratchadaphiseksomphot Endowment Fund). The funders with chronic kidney disease. J Vet Intern Med. 2007;21(5):906–16.
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publication fee. recommended? J Feline Med Surg. 2013;15(1_suppl):15–27.
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NC; Laboratory test: CT and CB; Analysis and interpretation of data: CT, SS, CB changes associated with disease. J Feline Med Surg. 2012;14(11):794–803.
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the Chulalongkorn University Animal Care and Use Committee (CU-ACUC), 15(10):849–57.
Faculty of Veterinary Science, Chulalongkorn University (Protocol number: 18. Soylemezoglu O, Wild G, Dalley AJ, MacNeil S, Milford-Ward A, Brown CB, El
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Consent for publication 19. Teppo AM, Törnroth T, Honkanen E, Grönhagen-Riska C. Urinary amino-
Written informed consent was obtained from the cats’ owners for terminal propeptide of type III procollagen (PIIINP) as a marker of interstitial
publication of this manuscript. fibrosis in renal transplant recipients. Transplantation. 2003;75:2113–9.
20. Ix JH, Biggs ML, Mukamal K, Djousse L, Siscovick D, Tracy R, Katz R, Delaney
Competing interests JA, Chaves P, Rifkin DE, Hughes-Austin JM, Garimella PS, Sarnak MJ, Shlipak
The authors declare that they have no competing interests. MG, Kizer JR. Urine collagen fragments and CKD progression—the
cardiovascular health study. J Am Soc Nephrol. 2015;26:2494–503.
Publisher’s Note 21. Glińska-Suchocka K, Orłowska A, Jankowski M, Kubiak K, Spużak J. Serum
Springer Nature remains neutral with regard to jurisdictional claims in concentrations of PIIINP aminopeptide in dogs with liver fibrosis. Pol J Vet
published maps and institutional affiliations. Sci. 2016;19(2):365–9.
22. Heikkilä HP, Krafft E, Jespers P, McEntee K, Rajamäki MM, Clercx C.
Author details Procollagen type III amino terminal propeptide concentrations in dogs with
1
Department of Veterinary Physiology, Faculty of Veterinary Science, idiopathic pulmonary fibrosis compared with chronic bronchitis and
Chulalongkorn University, Bangkok, Thailand. 2Department of Veterinary eosinophilic bronchopneumopathy. Vet J. 2013;196(1):52–6.
Surgery, Faculty of Veterinary Science, Chulalongkorn University, 39 23. Hezzell MJ, Boswood A, Chang YM, Moonarmart W, Elliott J. Associations
Henri-Dunant Road, Wangmai, Pathumwan, Bangkok 10330, Thailand. among serum N-terminal procollagen type III concentration, urinary
aldosterone-to-creatinine ratio, and ventricular remodeling in dogs with
Received: 24 July 2018 Accepted: 29 January 2019 myxomatous mitral valve disease. Am J Vet Res. 2012;73(11):1765–74.
24. Lin HY, Lee YL, Lin KD, Chiu YW, Shin SJ, Hwang SJ, Chen HC, Hung CC.
Association of renal elasticity and renal function progression in patients
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