June 2022

Medical Mass Spectrometry Vol. 6 No. 1

Research Paper

Urinary kidney injury molecule-1 as early diagnostic marker
of chronic kidney disease in cats
1

2

2

3

Hiroto Maeda , Kurumi Igarashi , Mako Ishidoya , Mamoru Satoh ,4

3

2

Toshifumi Watanabe , Fumio Nomura , Kazuyuki Sogawa *Maeda Veterinary Hospital, 1‒4‒8 Yamatemachi, Tomakomai, Hokkaido 053‒0851, Japan
Department of Biochemistry, School of Life and Environmental Science, Azabu University,
1‒17‒71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252‒5201, Japan
3
Divisions of Clinical Mass Spectrometry and Clinical Genetics, Chiba University Hospital,1‒8‒1 Inohana, Chuo-ku, Chiba, Chiba 260‒8670, Japan
4
Veterinary Teaching Hospital, Azabu University, 1‒17‒71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252‒5201, Japan
1

2

Abstract Chronic kidney disease (CKD) is a common disorder and cause of death in cats. In the classiﬁcation proposedby the International Renal Interest Society (IRIS), stage 1 and 2 CKD are diﬃcult to diagnose accurately using markers,
in comparison with normal controls. We recently described a simple and highly reproducible tandem mass tag labelling
method for identifying potential disease-marker candidates among low-abundance urine proteins. In the current study, urine
samples were obtained from 90 normal control cats as the control group and from 50 cats with CKD (stage 1). To identify
new urine biomarkers for CKD, two pool urine samples (normal controls and CKD stage 1 cats) were diﬀerentially labelledwith TMT, subjected to analysis using SDS-PAGE, digested with trypsin and subjected to analysis using LC-MS/MS.
Kidney injury molecule-1 (KIM-1) was identiﬁed as a protein with higher levels in cats with CKD (stage 1). An ELISA ofurine KIM-1 showed within-run (3.2‒4.5%) and between-day (3.4‒4.8%) reproducibility. Urine KIM-1 levels measured
with this assay were signiﬁcantly greater in CKD (stage 1) cats than in normal cats (63.7±10.7 vs. 35.7±9.7 μg/g Cre, p＜0.001). These results indicate that KIM-1 may be useful as a complementary marker with p-Cre and BUN for detection of
CKD (stage 1) in cats.
Key words: cat, chronic kidney disease, kidney injury molecule-1, urine

Introduction

progression of renal dysfunction, which leads to prolonga-

Feline renal diseases are increasingly common in veteri-2)

tion of survival and improvement of QOL . However, there

1)nary practice . It is important to diagnose and identify theare only a few reports on this area in clinical veterinary

pathological basis of renal dysfunction accurately at an

medicine .

3)

In daily clinical practice, plasma creatinine (p-Cre) andearly stage, since early diagnosis and treatment can delay

serum symmetric dimethylarginine (serum SDMA) are* Corresponding author

used as markers based on the stage classiﬁcation criteria of

Kazuyuki Sogawa

4)

the International Renal Interest Society (IRIS) (Table 1).

Department of Biochemistry, School of Life and Environmental Science, Azabu University, 1‒17‒71 Fuchinobe, Chuo-ku,

However, many of these markers have low speciﬁcity for

Sagamihara, Kanagawa 252‒5201, Japan
Tel: ＋81‒42‒769‒1924, Fax: ＋81‒42‒769‒1924

renal function, and there is a blind area without abnormal

Received: September 27, 2021. Accepted: October 28, 2021.
Epub December 10, 2021.

normal status to Stage 1 in the IRIS classiﬁcation for early
diagnosis of renal function. However, it is diﬃcult to evalu-

DOI: 10.24508/mms.2022.06.002

ate a mild decrease of renal function in the early phasevalues despite development of mild renal dysfunction. It isespecially important to evaluate the transitional period fromE-mail: sogawa@azabu-u.ac.jp

36June 2022

Table 1.

Medical Mass Spectrometry Vol. 6 No. 1

Staging of CKD based blood creatinine andSDMA concentrations

Stage

1

2

3

4

Blood creatinine(mg/dL)＜1.6

1.6‒2.8

2.9‒5.0＞5.0SDMA(μg/dL)

＜18

18‒25

26‒38

＞38

Table 2.

Variablesusing the available markers in clinical practice. In stage 1
there may not be any reduction in GFR or the surrogate
markers of GFR despite the fact there is active disease

Stage I

Stage IIAge (Mean±s.d.)6.8±3.6

6.7±3.3

6.8±3.6

Sex (male, female)

90 (43, 47)

50 (24, 26)

10 (5, 5)

Blood urea nitrogen(mg/dL)(Mean±s.d.)p-Creatinine (mg/dL)(Mean±s.d.)24.1±5.7

25.6±6.8

26.8±5.9

0.96±0.23

1.20±0.23

1.99±0.24

Urinary protein tocreatinine ratio(Mean±s.d.)techniques is stilllimited, but recently there have been sig5‒7)

CKD group

Controlgroup

Urine speciﬁc gravity 1.053±0.006 1.051±0.005 1.045±0.016(Mean±s.d.)ongoing in the kidney.
In veteri-nary medicine, the application of proteomics
niﬁcant eﬀorts to study the urine proteome in cats

Clinical characteristics and biochemical variables of the control groups and CKD groups.

0.13±0.04

0.15±0.05

0.17±0.06

In the present study, we employed the TMT method togenerate comparative protein proﬁles of urine samples
obtained from normal controls and CKD stage 1 cats. Fur-

2.8 mg/dL in stages 1 and 2, based on the IRIS guidelines.
The diagnosis of cats with stage 1 CKD was made if p-Cre

thermore, we compared the urine levels of candidate pro-was ＜1.6 mg/dL, while urine concentrating ability had

teins to evaluate the ability to discriminate between normal
controls and CKD cats. Here, we identify novel urine bio-

been lost, other disorder for decreased urine concentrating

markers that are potentially superior to classical markers

consistent with CKD (e.g., small kidney size and poor orfor early and speciﬁc detection of CKD stage 1 cats.

absent renal corticomedullary diﬀerntiation) . Urine wasability were excluded, and ultrasonographic changes were9)collected by catheterization. Samples were centrifuged atMaterials and Methods

1,190×g for 10 min (Kubota, Tokyo, Japan) and then storedat −80°
C for further use. All owners gave signed informed

Animals
Table 2 shows Clinical characteristics and biochemicalconsent to participation of their animal in the study.

variables of the control groups and CKD groups. Urine

Urine concentration

samples from 50 cats with stage 1 and 10 cats with stage 2chronic kidney disease (CKD) brought to Maeda Veterinary

Ultraﬁltration membrane sample preparation using the

Hospital between January 6, 2017 and February 20, 2018

Nanosep centrifugal devices (Pall Life Sciences, Ml, USA)
were used to process the urine samples. The Nanosep cen-(American Shorthair: 5, Chinchilla: 2, Mix: 53; 29 males
and 31 females aged 2‒13 years) were used in the study.

trifugal devices procedure was prepared according to the
manufacturer's instructions. Urine (500μL) were trans-

Urine samples from 90 cats aged 2‒13 years (Scottish Fold:
4, American Shorthair: 2, Mix: 84; 43 males and 47
females) were used as normal controls. Regarding the diag-ferred to a sample reserver (Pall Life Sciences, USA), were

nostic criteria, cats clinically diagnosed as normal based on

The ﬁnal volume was 20μL.

centrifuged at 14,000×g for 20 min (Kubota, Tokyo, Japan).

serum biochemistry and ultrasonography tests were deﬁned

Tandem mass tag (TMT) labellingas normal cats, and cats showing clinical signs of CKD inblood chemistry were deﬁned as those with stage 1 CKD.

Reduction and alkylation were performed as describedCriteria for normal urinalysis were urine speciﬁc gravity

previously . TMTsixplex Isobaric Label Reagent Set(USG) ＞1.030, urinary protein to creatinine ratio (UPC)8)≤ 0.2, and a negative bacteriologic urine culture . Diagno-(Pierce, ID, USA) was used according to the manufacturer's
instructions. Tubes containing the diﬀerent isobaric chemi-

sis of CKD was made prior to inclusion in the study, based

cal tags (0.8 mg each) were added to 41 μL of anhydrous

on clinical and laboratory ﬁndings. After stabilization, cats8)
were classiﬁed into 4 groups with p-Cre of ＜1.6 and 1.6‒

acetonitrile (ACN) and dissolved for 5 min with occasional

10)

vortexing at room temperature. TMT solution (20μL) was
37

June 2022

Medical Mass Spectrometry Vol. 6 No. 1

added to each tube and allowed to react at room tempera-

ter and analyzed with parameters according to previous

ture for 60 min. A volume of 4μL of 5% hydroxylamine

research .

was added to each sample, and the mixture was incubated

The database search engine (Proteome discoverer;
Thermo Scientiﬁc) was used to identify and quantify pro-

12)

10)

for 15 min to quench the reaction . Samples were ﬁnallypooled and lyophilized. Analyses were performed for sam-teins from the mass, tandem mass and reporter ion spectra

ples of 20μL of concentrated pooled urine from normal
controls (n＝8) (TMT6-126 and TMT6-128) and from stageof peptides. Peptide mass data were matched by searching

1 CKD cats (n＝8) (TMT6-127 and TMT6-129).

the International Protein Index database (IPI, European
Bioinformatics Institute) and the cat KIM-1 sequence. The

SDS-PAGE analysis and in-gel digestion of proteins

database search parameters employed were, peptide masstolerance＝2 p.p.m. and fragment tolerance＝0.6 Da. Further-

Lyophilized samples were dissolved in PAGE sample
buﬀer (15μL) were analyzed by SDS-PAGE (Perfect NT

more, the enzyme parameter was set to trypsin, allowing up

Gel W; DRC Co. Ltd., Tokyo, Japan, 10‒20% acrylamide,oxidation, and cysteine alkylation. The minimum criteria of

20 wells). The gel was stained with Coomassie brilliant

protein identiﬁcation were ﬁltered with Xcorr vs charge

blue (PhastGel Blue R; GE Healthcare, Little Chalfont,state and set as false discovery rate (FDR)＜1%.

to one missed cleavage variable modiﬁcations, methionine

UK).

ELISA

The whole lane was cut into 20 pieces. The gel was cut
into small pieces, destained in 50% ACN/50 mM NH4HCO3
and washed with deionized water. Gel pieces were dehy-

Immunogens for development of anti-KIM-1 antibodies:
Recombinant KIM-1 was made by Fujiﬁlm Wako Puredrated in 100% ACN for 15 min and dried in a SpeedVac

Chemical Corp (Osaka, Japan). Synthetic peptides of 20
amino acids (Toray Research Center, Tokyo, Japan) corre-

Evaporator (Wakenyaku, Kyoto, Japan) for 45 min. The
pieces were rehydrated in 10‒30μL of 25 mM Tris-

sponding to the sequences of the KIM-1 N-terminus (KIM-1
N peptide) and C-terminus (KIM-1 C peptide) coupled toHCl/20% ACN containing 25 ng/L trypsin (Trypsin

keyhole limpet hemocyanin (KLH) were obtained from Sigma-Aldrich Japan (Tokyo, Japan). The peptide-KLH conju-

sequence grade, Roche, Basel, Switzerland) for 45 min.
After removal of unabsorbed solution, the gel pieces wereincubated in 10‒20 μL of 50 mM Tris-HCl/20% ACN for

gates were dissolved in distilled water and used as antigens

20 h at 37°
C. The solution containing digested protein fragments was transferred to a new tube, and peptides remain-for preparation of monoclonal antibodies.
Immunization and establishment of hybridoma cell lines:
KIM-1 N and C peptides (50 mg at 1 mg/mL in PBS) were

ing in the gel were extracted with 5% formic acid/50%
11)

ACN for 20 min at room temperature .

used for immunization of BALB/c mice. Hybridoma cell
13)
lines KIM-1 N-02 and C-01 were established and anti-

Protein identiﬁcation and quantiﬁcation by LC-MS/
MS analysis

body isotypes were determined using a Mouse Monoclonal

The tryptic digested peptides were injected into a trap

To obtain pure monoclonal antibodies on a large scale,column (C18, 5μm, 0.3×5 mm; DIONEX, Sunnyvale, CA,

BALB/c mice were initially stimulated with 1.0 mL pristineUSA) and an analytical column (C18, 3μm, 0.075×(Sigma Aldrich Japan) and then inoculated 2 weeks later.
Monoclonal antibodies were puriﬁed as described else-

Antibody Isotyping Test Kit (AbD Serotec, Oxford, UK).

120 mm; Nikkyo Technos, Tokyo, Japan), which was

13)

attached to the Ultimate 3000 (DIONEX). The ﬂow rate of−1
the mobile phase was 300 nL min . The solvent composi-

where .

tion of the mobile phase was programmed to change in 120min cycles, with varying mixing ratios of solvent A (2% v/v

examine the speciﬁcity of the antibodies, recombinant
KIM-1 (10 ng) and urine samples of cats with CKD stage 1

acetonitrile (ACN) and 0.1% v/v formic acid) to solvent B(90% v/v ACN and 0.1% v/v formic acid) as described pre-(5 μL concentrated urine) were separated by SDS-PAGE ona 10‒20% gradient gel (DRC, Tokyo, Japan), and thenWestern blot analysis using anti-KIM-1 antibodies: To

12)viously . Puriﬁed peptides were introduced from HPLC to
the LTQ-Orbitrap XL (Thermo Scientiﬁc, San Jose, CA,

transferred onto a PVDF membrane (Millipore, Billerica,
MA, USA). To minimize nonspeciﬁc binding, the mem-

USA), a hybrid ion-trap Fourier transform mass spectrome-branes were incubated with Blocking One (Nacalai Tesque,38

June 2022

Medical Mass Spectrometry Vol. 6 No. 1

Other procedures

Kyoto, Japan). After the membrane was washed three times
with PBST (PBS buﬀer including 0.05% Tween-20), theCreatinine was measured enzymatically with creatinine
deiminase using a Fuji DRI-CHEM Slide CRE-PIII kit

membranes were incubated with anti-KIM-1 N peptide antibody or anti-KIM-1 C peptide antibody as primary antibod-(FujiFilm Medical, Tokyo, Japan). Blood urea nitrogen(BUN) was measured using a N-Assay BUN-L Nittobo

ies for 1 h at room temperature. The membrane was washedagain three times with PBST and then incubated with a sec-

D-Type kit (Nittobo Medical Co., Ltd., Tokyo, Japan).
Numerical data are presented as the mean±standard devia-

ondary rabbit anti-mouse immunoglobulin/HRP (DakoJapan, Tokyo, Japan) for 1 h at room temperature. The reac-tion (SD).

tive antibodies were visualized by staining with Pierce
Western Blotting substrate (Thermo Fisher Scientiﬁc).

Statistical analysis

Immobilization of antibodies on a polystyrene microtiterplate: The anti-KIM-1 C peptide antibody dissolved in PBS

Statistical analysis was performed using IBM SPSS
Statistics 19 (SPSS Inc., Chicago, IL, USA). For non-buﬀer was dispensed into a 96-well polystyrene microtiterplate (Thermo Fisher Scientiﬁc) at 0.5 mg/well and incu-

parametric data, the diﬀerences between two groups wereanalysed using the Mann‒Whitney U-test. P-values＜0.05

bated for 1 day at 4°
C. The plate was washed three times
with PBS containing 0.05% Tween-20. The microtiter plate

were considered statistically signiﬁcant. Receiver operating

was coated with 20% NOF102 containing 10% sucrose forthe sensitivity, speciﬁcity, and respective areas under
the curves (AUC). Levels of urinary KIM-1 was expressedcharacteristic (ROC) curves were constructed to assess

1 day at 4°C.

in the Creatinine-corrected urinary KIM-1 (μg/g Cre) levels.

ELISA conditions: Recombinant KIM-1 was diluted with
PBS buﬀer for calibration. After washing the microtiterplate with PBST, 100-μL aliquots of 10-times diluted urine

Results
Identiﬁcation of candidate proteins by comparing theurine of normal controls and CKD stage 1 cats using
the TMT method

samples were added in duplicate to wells. The plates wereincubated at room temperature for 1 h and then washedthree times. HRP-conjugated anti-KIM-1 N antibody in
PBST (100 μL) was added to each well and the plate was

To identify new urine biomarkers for CKD, two poolincubated at room temperature for 30 min. The plate wasurine samples (normal controls and CKD stage 1 cats) were

washed three times, and then 100 μL of TMB solution(Wako Pure Chemical Industries) was added. After incuba-

diﬀerentially labelled with TMT, subjected to analysis
using SDS-PAGE, digested with trypsin and subjected totion at room temperature for 10 min, 100 μL of stop solution

analysis using LC-MS/MS (Fig. 1). Analyzing urinary protein by SDS-PAGE after TMT labeling can accurately deter-

was added and the absorbance at 450 nm was measured.
Sensitivity, Precision, and Accuracy: Blank controls at aconcentration of 0 ng/mL KIM-1 in 8 replicates were usedmine the molecular weight and eliminate urinary protein

to determine the sensitivity and the detection limit. Two different concentrations of the KIM-1 (2 ng/mL, 12 ng/mL) in

was labelled with TMT with reporter ion at m/z＝126 ordegradation products. Of these, normal control pool sample
128, and CKD stage 1 cat pool sample was labelled with
TMT with reporter-ion at m/z＝127 or 129. After comparing

eight replicates were used to determine the within-assay
variation. Two diﬀerent concentrations of the KIM-1 (2 ng/mL,

the proﬁles of proteins by LC-MS/MS using an LTQ

12 ng/mL) in 5 diﬀerent days were used to determine thebetween-assay variation. Precision was evaluated by within/

Orbitrap XL mass spectrometer, 47 proteins with uniquebetween-assay coeﬃcients of variation (CVs) (%), withwithin-assay CV ＜10% and between-assay CV ＜20%were ﬁve proteins with urine levels in stage 1 CKD catsthat were more than two times than the level in normal con-being acceptable. Accuracy was evaluated by recovery. The
observed and expected concentrations of KIM-1 in with-trols (Table 3). The protein with the largest elevation was
identiﬁed as KIM-1.

peptide sequences were found (Table 3, Table 4). Therein-assay were used to analyze the recovery. A recovery of
75‒125% was acceptable.

Establishment and characterization of ELISA for measuring KIM-1
Anti-KIM-1 N peptide antibody (Fig. 2A) and anti-KIM-1

39

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Medical Mass Spectrometry Vol. 6 No. 1

Fig. 1.
Table 3.

Identiﬁcation of candidate proteins using TMT labelling and LC-MS/MS analyses.

Urine proteins detected at higher levels in CKD stage 1 cats compared to normal controls in proteome analysis

Database
Accession No.

ID

Coverage(%)

Number of matching
peptidesTMT rate(127/126)

TMT rate(129/128)

5

3.4

3.6

gi 757173465

Kidney injury molecule-1

12

Q29487(Hepatitis A virus cellular receptor 1)
Prostaglandin-H2 D-isomerase

14

6

2.6

2.7

Q8I034

Carboxylesterase 5A

9

6

2.5

2.4

O62763

Acetylcholinesterase

12

6

2.3

2.2

E3UTY9Haptoglobin
Angiotensin-converting enzyme 2

16

2

2.2

2.0

14

2

1.8

2.0

Dipeptidyl peptidase 4
Beta-lactoglobulin-2

8

5

1.6

1.9

P21664

10

3

1.6

1.9

P07405

Hemoglobin subunit alpha

15

4

1.6

1.8

P49064

12

4

1.5

1.7

10

4

1.5

1.7

D5MTH1

Serum albumin
Beta-glucuronidase
Cystatin-C

9

2

1.5

1.6

P79171

Aminopeptidase N

11

5

1.4

1.6

P13369Macrophage colony-stimulating factor 1 receptor
Stromal cell-derived factor 1

10

2

1.4

1.6

9

3

1.4

1.5

13

2

1.4

1.5

Q95ND4

Beta-galactosidase
Pro-epidermal growth factor

15

6

1.3

1.5

Q8WNR9Cystatin-A

13

4

1.3

1.4

Q76B49

13

5

1.3

1.4

Q5MGS7

CD63 antigen
Beta-2-microglobulin

15

6

1.3

1.4

O46432

Lysosomal alpha-mannosidase

14

2

1.3

1.4

Q6SA95

Coagulation factor IX

15

2

1.2

1.4

Q56H28Q9N2I7

O97524

O62657
O19015Q2WGK2Junctional adhesion molecule A

9

4

1.2

1.3

Q2MHN1

15

2

1.2

1.3

P58727Ferritin light chain
Toll-like receptor 4

7

4

1.2

1.3

P49614

Beta-hexosaminidase subunit beta

12

5

1.2

1.3

P40239CD9 antigen

9

2

1.2

1.3

M5AXY1Retinol binding protein 4

5

3

1.2

1.3

M3W955

Apolipoprotein A-IV

8

2

1.2

1.3

Q7YRU4

6

3

1.1

1.3

P33687

Malate dehydrogenase, cytoplasmic
Beta-lactoglobulin-1

10

5

1.1

1.2

P14450Fibrinogen alpha chain (Fragment)

9

2

1.1

1.1

40

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Medical Mass Spectrometry Vol. 6 No. 1

Table 4.

Urine proteins detected at lower levels in CKD stage 1 cats compared to normal controls in proteome analysis

Database
Accession No.

ID

Coverage(%)

Number of matching
peptidesTMT rate(127/126)

TMT rate(129/128)

M3W551

Antioxidant 1 copper chaperone

14

8

0.9

0.8

M3W8S0

Ezrin

7

4

0.9

0.8

A0A2I2U4B5

Nucleoside diphosphate kinase

9

8

0.9

0.7

A0A2I2UZE5

11

5

0.9

0.7

M3X5I6

Peroxiredoxin 1SH3 domain-binding glutamic acid-rich-like protein

9

4

0.9

0.7

M3X9A1

Triosephosphate isomerase

5

5

0.9

0.7

M3WM96

Annexin

11

3

0.8

0.7

M3VWL2Fructose-bisphosphate aldolase
Glyceraldehyde-3-phosphate dehydrogenase

7

4

0.8

0.7

Q9N2D5

9

3

0.8

0.7

P37155

Lysozyme C

6

3

0.8

0.7

M3X166

Phosphoglycerate kinase

2

3

0.8

0.7

A0A2I2UPH5

10

3

0.8

0.7

M3WPG6

Superoxide dismutase [Cu‒Zn]
Apolipoprotein A-I

3

2

0.7

0.7

M3VZP6

Gelsolin

7

2

0.7

0.6

A0A2I2UJB3Radixin

4

2

0.7

0.6

Fig. 2.

Western blotting analysis.

Immunoreactive bands were observed for KIM-1 when recombinant and urine KIM-1 were incubated with anti-KIM-1 N
peptide antibody (A) or anti-KIM-1 C peptide antibody (B). Lane 1: recombinant KIM-1 (10 ng). Lane 2: urine samples ofcats with CKD stage 1 (5 μL concentrated urine).

C peptide antibody (Fig. 2B) recognized recombinant
KIM-1 and urine samples from CKD stage 1 cats, respec-

determined with eight replicates of each sample.
Between-assay CVs were determined based on assays per-tively.

formed on 5 diﬀerent days (two replicates of each sample
per day). The within-run CV was 3.2‒4.5% and theRange, dilution analysis and detection limit: A standard

between-run CV was 3.4‒4.8%.

curve was drawn based on the colorimetric intensity ofdiluted recombinant KIM-1 to establish the relationship ofintensity with KIM-1 concentration (Fig. 3). The working

Interference: Interference was assessed in samples containing 5.0 ng/mL KIM-1. Potential interference materials

range of the assay was 0‒50 ng/mL and the assay gave lin-

were added to urine at various concentrations. There was

ear results from 0 to 50 ng/mL (y＝1.8292x＋0.0498, r2＝

no substantial interference from hemoglobin (up to
5,000 mg/L), free bilirubin (up to 207 mg/L), ditaurobiliru-

0.9999, p＜0.0001). The detection limit was estimated byassaying the zero concentration eight times, and was
deﬁned as the KIM-1 zero concentration＋3SD. The limitbin (up to 204 mg/L), chyle (up to 1,400 formazine turbid-

was found to be 0.32 ng/mL.
Within-run and between-run reproducibility: The preci-(up to 500 mg/L), and rheumatoid factor (up to 500 U/L).

ity units, equal to 1,176 mg/L triglyceride), ascorbic acid
Recovery test: To evaluate recovery in the ELISA, 0.5
and 5.0 ng/mL of synthetic KIM-1 were added to pooledsion of the assay was determined using two KIM-1 concentrations of 2.0 and 12.0 ng/mL. Within-assay CVs wereurine (2.8 ng/mL). The percentage recovery ranged from41

June 2022

Fig. 3.

Medical Mass Spectrometry Vol. 6 No. 1

Standard curves for urine KIM-1 in the ELISA.

Colorimetric intensity and urine KIM-1 concentration
were related in the range of 0‒50 ng/mL. Six concentrations of urine KIM-1 were determined by ELISA.

Fig. 5.

Urine KIM-1 is more eﬀective than p-Cre and
BUN for early detection of stage 1 CKD.
ROC analyses were performed for the serum levels ofurine KIM-1, p-Cre and BUN in stage 1 CKD and normal cats. The AUCs were 0.964 for urine KIM-1, 0.698
for p-Cre, and 0.554 for BUN.

stage 1 CKD cats than in normal cats (63.7±10.7 vs. 35.7±
9.7 μg/g Cre, p＜0.001; Mann‒Whitney U-test). Urine
KIM-1 was signiﬁcantly higher in stage 2 CKD cats than instage 1 CKD cats (89.1±19.3 vs. 63.7±10.7 μg/g Cre, p＜0.001; Mann‒Whitney U-test). Receiver operator characteristic (ROC) curves were constructed to evaluate the urine
KIM-1, serum creatinine, and serum BUN cut-oﬀs for distinguishing stage 1 CKD cats from normal cats. The respective areas under the ROC curve (AUCs) were 0.964 forurine KIM-1, 0.698 for serum creatinine, and 0.554 forserum BUN. These results suggest that urine KIM-1 may be
a better diagnostic stage 1 CKD biomarker than serum creFig. 4.

Urine KIM-1 levels in normal controls and CKD
stage 1 cats.

atinine and BUN (Fig. 5). The ROC curves showed that the
optimal diagnostic cut-oﬀ value was 49.4 ng/mL for urine

Urine KIM-1 was signiﬁcantly higher in CKD stage 1cats (63.7±10.7 μg/g Cre) compared with normal cats
(35.7±9.7 μg/g Cre). p＜0.001 by Mann‒Whitney U-test.
Urine KIM-1 was signiﬁcantly higher in stage 2 CKD
cats than in stage 1 CKD cats (89.1±19.3 vs. 63.7±
10.7 μg/g Cre, p＜0.001; Mann‒Whitney U-test).

KIM-1. Moreover, the AUC for urine KIM-1 was 0.964,
with a sensitivity of 90.0% and a speciﬁcity of 90.5%. The
Pearson correlation coeﬃcients of urinary KIM-1were
0.096 for BUM, and 0.322 for p-Cre, respectively.

Discussion

96.2 to 103.9%.

In this study, SDS-PAGE and proteomics analyses wereused to detect Beta-2-microglobulin and KIM-1 in urine

KIM-1 levels are increased in the urine of cats with
CKD
Urine KIM-1 levels were measured in stage 1 CKD catsfrom CKD stage 1 cats and normal controls. The proteinwith the largest elevation was identiﬁed as KIM-1. KIM-1,(n＝50), stage 2 CKD cats (n＝10) and normal cats (n＝90).

which is also known as hepatitis A virus cell receptor 1

As shown in Fig. 4, urine KIM-1 was signiﬁcantly higher in(HAVCR1) and T cell immunoglobulin 1, is a renal tubular42

June 2022

Medical Mass Spectrometry Vol. 6 No. 1

transmembrane glycoprotein that is thought to function in
14,15). KIM-1 is known
cell-to-cell or cell-to-matrix adhesion

marker for CKD that is complementary to conventional

to be undetectable in normal kidney tissue but is expressedat very high levels in dediﬀerentiated proximal tubule epi-

useful for establishing the diagnostic signiﬁcance of urine
KIM-1 in CKD stage 1 cats.

thelial cells in human and rodent kidneys after ischemic or
16‒17). The urine KIM-1 concentration is cortoxic injuries

Conclusion

markers. The simple ELISA developed in this study was

Our results suggest that KIM-1 may be used as a marker
complementary to p-Cre and BUN for detection of CKD

related with acute kidney injury (AKI) severity and18)

decreases as kidney repair progresses .
Vaidya et al., have already constructed a sandwich(stage 1) in cats. We note that the study is limited by the

ELISA kit for KIM-1 detection in rat urine using two
17)
mouse monoclonal antibodies . The sandwich Kim-1

number of samples, and evaluation of more samples frommultiple facilities is required in a future study. The fact that
longitudinal follow-up of cats in stage 1 CKD would have

ELISA was to develop a sensitive quantitative urinary test
to identify renal injury in the rodent. Two mouse monoclo-

helped to determine those that have true CKD which is progressive vs. those still recovering from AKI when the sam-

nal antibodies were made against the puriﬁed ectodomain
of Kim-1, and these were used to construct a sandwich

ples were taken.

Kim-1 ELISA. The assay range of this ELISA was 50 pg/mL
to 5 ng/mL, with inter- and intra-assay variability of ＜10%.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientiﬁc

In addition, previous reports have shown that three
expressed feline KIM-1 transcript variants comprising 894,

Research (KAKENHI C: 19K07947) and a research project
grant awarded by Azabu University.

810, and 705 bp were identiﬁed in renal tissue, and theintensity of KIM-1 expression is increased proportionallyConﬂict of Interest. Immunohistochemistry of kidney sections identiﬁed KIM-1 in proxi-

with the severity in kidney-damaged cats

19,20)

There are no conﬂict of interest relevant to this article.

mal tubular cells of cats with positive urine immunoassay
References

20,21)

results, but a quantitative method has not previously

1) Syme HM, Fletcher MG, Bailey SR, Elliott J: Measure-been established. We have constructed a sandwich ELISA
kit for KIM-1 detection in cat urine using KIM-1 N peptide

ment of aldosterone in feline, canine and human urine. J

and KIM-1 C peptide antibodies. We developed an ELISA
that was satisfactory in terms of recovery (96.2‒103.9%)

2) King JN, Tasker S, Gunn-Moore DA, Strehlau G; BEN-

and within-run (3.2‒4.5%) and between-day (3.4‒4.8%)

RIC (benazepril in renal insuﬃciency in cats) Study

reproducibility. This assay was used to measure urine
KIM-1 as a potential biomarker to distinguish CKD stage 1

Group: Prognostic factors in cats with chronic kidney dis-cats from normal cats.
KIM-1 was identiﬁed by proteomics analysis, and such3) Tomura S, Saito K, Kawada K, Hirano C, Yanagi H, et al:

analyses have resulted in identiﬁcation of many new biomarkers, but most are not appropriate for clinical applica-

semiquantitative gold colloid dipstick method with thetion. The procedures for developing biomarkers include
comparison of disease and control groups by semiquantita-

mary Care 22: 37‒40, 1999.
4) Relford R, Robertson J, Clements C: Symmetric dimethy-

tive analysis (discovery phase) and selection of marker can-larginine: Improving the diagnosis and staging of chronic

didates (validation phase), followed by ELISA validation of

kidney disease in small animals. Vet Clin North Am Small

markers found with proteomics. Use of proteomics allows acomprehensive analysis of protein levels in vivo and rela-Anim Pract 46: 941‒960, 2016.

Small Anim Pract 48: 202‒208, 2007.

ease. J Vet Intern Med 21: 906‒916, 2007.
Measurement of microalbuminuria. Comparison of thequantitave turbidmetric immunoassay method. Jpn J Pri-

5) Lemberger SIK, Deeg CA, Hauck SM, Amann B, Hirmer
S, et al: Comparison of urine protein proﬁles in cats with-

tionships with disease.
Urine KIM-1 in CKD stage 1 cats (63.7±10.7 μg/g Cre)out urinary tract disease and cats with idiopathic cystitis,

was signiﬁcantly higher than that in normal cats (35.7±
9.7 μg/g Cre) (p＜0.001). This suggests that KIM-1 is a newbacterial urinary tract infection, or urolithiasis. Am J Vet
Res 72: 1407‒1415. 2011.
43

June 2022

Medical Mass Spectrometry Vol. 6 No. 1

the production of felinine, a putative pheromone precur-6) Jepson RE, Coulton GR, Cowan ML, Markwell P, Syme

sor. Chem Biol 13: 1071‒1079, 2006.

HM, et al: Evaluation of mass spectrometry of urinary
proteins and peptides as biomarkers for cats at risk of de-14) Ichimura T, Bonventre JV, Bailly V, Wei H, Hession CA,

veloping azotemia. Am J Vet Res 74: 333‒342, 2013.

et al: Kidney injury molecule-1 (KIM-1), a putative epi-

7) Ferlizza E, Campos A, Neagu A, Cuoghi A, Bellei E, etthelial cell adhesion molecule containing a novel immu-

al: The eﬀect of chronic kidney disease on the urine pro-noglobulin domain, is up-regulated in renal cells after in-teome in the domestic cat (Felis catus). Vet J 204: 73‒81,

jury. J Biol Chem 273: 4135‒4142, 1998.

2015.

15) Bailly V, Zhang Z, Meier W, Cate R, Sanicola N, et al:8) Board NKFKDOQIA: Clinical practice guidelines for

Shedding of kidney injury molecule-1, a putative adhe-chronic kidney disease: Evaluation, classiﬁcation, andsion protein involved in renal regeneration. J Biol Chem

stratiﬁcation IV: Deﬁnition and classiﬁcation of stages of

277: 39739‒39748, 2002.

chronic renal failure. Am J Kidney Dis 39: S46‒S75, 2002.

16) Han WK, Bailly V, Abichandani R, Thadhani R, Bonven-9) Debruyn K, Haers H, Combes H, Paepe D, Peremans K,

tre JV: Kidney Injury Molecule-1 (KIM-1): A novel bio-

et al: Ultrasonography of the feline kidney: Technique,marker for human renal proximal tubule injury. Kidney

anatomy and changes associated with disease. J Feline

Int 62: 237‒244, 2002.

Med Surg 14: 794‒803, 2012.

17) Vaidya VS, Ramirez V, Ichimura T, Bobadilla NA, Bon-

10) Sogawa K, Takano S, Iida F, Satoh M, Tsuchida S, et al:ventre JV: Urinary kidney injury molecule-1: A sensitive

Identiﬁcation of a novel serum biomarker for pancreaticquantitative biomarker for early detection of kidney tubu-

cancer, C4b-binding protein α-chain (C4BPA) by quanti-

lar injury. Am J Physiol Renal Physiol 290: F517‒F529,

tative proteomic analysis using tandem mass tags. Br J

2006.

Cancer 115: 949‒956, 2016.

18) Bonventre JV: Kidney injury molecule-1 (KIM-1): A spe-

11) Sogawa K, Kodera Y, Satoh M, Kawashima Y, Umemura

ciﬁc and sensitive biomarker of kidney injury. Scand J

H, et al: Increased serum levels of pigment epithelium-de-Clin Lab Invest Suppl 24: 178‒183, 2008.

rived factor by excessive alcohol consumption: Detection

19) Bland SK, Côté O, Clark ME, DeLay J, Bienzle D: Char-and identiﬁcation by a three-step serum proteome analy-

acterization of kidney injury molecule-1 in cats. J Vet In-

sis. Alcohol Clin Exp Res 35: 211‒217, 2011.

tern Med 28: 1454‒1464, 2014.

12) Tsuchida S, Satoh M, Kawashima Y, Sogawa K, Kado S,20) Bland SK, Schmiedt CW, Clark ME, DeLay J, Bienzle D:

et al: Application of quantitative proteomic analysis using

Expression of kidney injury molecule-1 in healthy andtandem mass tags for discovery and identiﬁcation of nov-

diseased feline kidney tissue. Vet Pathol 54: 490‒510,

el biomarkers in periodontal disease. Proteomics 13:

2017.

2339‒2350, 2013.

21) Bland SK, Clark ME, Côté O, Bienzle D: A speciﬁc im-13) Miyazaki M, Yamashita T, Suzuki Y, Saito Y, Soeta S,

munoassay for detection of feline kidney injury molecule
1. J Feline Med Surg 21: 1069‒1079, 2019.

et al: A major urinary protein of the domestic cat regulates

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