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O R I G I N A L R E S E A R C H
Characterization of the canine urinary proteomeLaura E. Brandt1, E. J. Ehrhart1,2, Hataichanok Scherman4, Christine S. Olver1, Andrea A. Bohn1,Jessica E. Prenni3,4
1Department of Microbiology, Immunology and Pathology; 2Animal Cancer Center; 3Department of Biochemistry and Molecular Biology; and4Proteomics and Metabolomics Facility, Colorado State University, Fort Collins, CO, USA
KeyWords
Biomarker, canine, exosome, proteomics,
urine
Correspondence
Jessica E. Prenni, Department of Biochemistry
and Molecular Biology, and Proteomics and
Metabolomics Facility, 2021 Campus Delivery,
Colorado State University, Fort Collins, CO
80523, USA
E-mail: [email protected]
DOI:10.1111/vcp.12147
Background: Urine is an attractive biofluid for biomarker discovery as it is
easy and minimally invasive to obtain. While numerous studies have
focused on the characterization of human urine, much less research has
focused on canine urine.
Objectives: The objectives of this study were to characterize the uni-
versal canine urinary proteome (both soluble and exosomal), to deter-
mine the overlap between the canine proteome and a representative
human urinary proteome study, to generate a resource for future
canine studies, and to determine the suitability of the dog as a large
animal model for human diseases.
Methods: The soluble and exosomal fractions of normal canine urine were
characterized using liquid chromatography tandem mass spectrometry
(LC-MS/MS). Biological Networks Gene Ontology (BiNGO) software was
utilized to assign the canine urinary proteome to respective Gene Ontology
categories, such as Cellular Component, Molecular Function, and Biologi-
cal Process.
Results: Over 500 proteins were confidently identified in normal canine
urine. Gene Ontology analysis revealed that exosomal proteins were lar-
gely derived from an intracellular location, while soluble proteins included
both extracellular and membrane proteins. Exosome proteins were
assigned to metabolic processes and localization, while soluble proteins
were primarily annotated to specific localization processes. Several proteins
identified in normal canine urine have previously been identified in
human urine where these proteins are related to various extrarenal and
renal diseases.
Conclusions: The results of this study illustrate the potential of the dog as
an animal model for human disease states and provide the framework for
future studies of canine renal diseases.
Introduction
In recent years, many researchers have utilized mass
spectrometry to characterize the human urinary prote-
ome1–7, and several of these studies have revealed
promising potential biomarkers specific for both renal
disease and extrarenal pathologic conditions.2–4,8–12
Urine is an attractive biofluid for biomarker discovery
as it is easy to obtain byminimally invasive procedures.
In addition, it is abounding with proteins, making
it ideal for diagnostic testing. The application of pro-
teomics for the analysis of canine urine so far has been
limited. Most studies have been limited in scope
and have utilized technologies that focus on the
identification of select proteins, the presence of which
has been found to differ according to environmental
states or disease.2,3,13,14 For example, two-dimensional
gel electrophoresis followed by mass spectrometry was
used to identify specific proteins related to tubulointer-
stitial injury in a canine model of progressive glomeru-
lar disease.15 In another study, surface-enhanced laser
desorption/ionization time-of-flight mass spectrome-
try was used to perform protein-level profiling of urine
from dogs with and without renal disease. 14 However,
none of these studies provided a comprehensive
proteomic profile of canine urine.
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Comprehensive proteomic analysis of any bio-
fluid, including urine, is difficult due to the qualitative
and quantitative complexity and wide range of pro-
teins. Fortunately, the analysis of urine can be focused
by targeting exosomes, which, due to their unique
acquisition of vesicular membranes and liquid
contents, provide very specific information about the
cells from which they originate. Exosomes are formed
by a stepwise process. When plasma membrane pro-
teins are endocytosed into a cell, endocytic vesicles are
generated. By inward budding, these endocytic vesi-
cles become multivesicular bodies (MVB), which con-
tain their own vesicles. After moving back to the cell
surface, the MVB bind to the cellular plasma mem-
brane, and release their internal vesicles, the exo-
somes, outside of the cell. Exosomes have a
cytoplasmic-side inward orientation, similar to intact
cells, and are thus loaded with specialized information
about their originating cells.16 They contain both
membranous and cytoplasmic constituents and are
generally excreted from cells for specific reasons, such
as cell-to-cell signaling or to act as membrane transport
proteins.16
The urinary exosomal population is thought to be
comprised largely of exosomes from renal and other
urinary tract cells. However, exosomes are also found
in serum and, due to their generally small size, can be
freely filtered by the glomerulus into the urinary
space.5 Theoretically, if a nonrenal disease causes an
increase of specific exosomes in serum, their proteins
may be found in greater abundance in urine. There-
fore, the analysis of the urine subproteome has the
potential to provide important information about the
urinary tract as well as systemic health, even though it
may be impossible to determine the actual origin of
urinary exosomes.
Thus, the goal of our current study was to adapt
proteomic methods established for the investigation of
human urine to canine urine, to characterize both ex-
osomal and soluble proteins. A comprehensive com-
parison based on protein annotation and gene
ontology was performed between our results and pub-
lished data of the human urinary proteome.1 Several
proteins previously identified in human urine and
associated with various disease states, including extra-
renal disease, renal disease, systemic hypertension,
and neoplasia, were also identified in canine urine.17–
26 While making an association between the observa-
tions of these proteins in canine urine with similar
disease processes requires further investigation, their
presence illustrates the potential for veterinary diag-
nostics as well as the use of canine models for human
conditions using investigative proteomic techniques.
Materials andMethods
Canine urine collection and protein concentration
Inclusion criteria required that dogs be between one
and 10 years of age, clinically normal, not taking any
medications or supplementations, and fed a commer-
cial adult dog food diet. Clinically normal was defined
as a healthy animal with unchanged biologic and social
functions in its known environment, and no abnor-
malities on physical examination. The health of all
dogs was further evaluated by routine CBC, including
evaluation of blood film, serum chemistry profile, uri-
nalysis, and urine protein to creatinine ratio (UPC).
Protocols for animal handling and sample collection
were reviewed and approved by the Colorado State
University Animal Care and Use Committee (ACUC).
Fifty milliliters of urine were collected by free
catch into sterile urine collection cups from all dogs.
A protease inhibitor cocktail (Sigma-Aldrich, St.
Louis, MO, USA) was added at a ratio of 1:10 to each
sample immediately after collection to avoid protein
degradation.27 Samples were immediately frozen to
�80°C, they were stored until all samples were col-
lected and could be analyzed in batch within
12 months of collection.27 Samples were thawed in
cool water and then kept on ice during processing.
After vortexing to reconstitute particles that may
have settled during the freezing process, all samples
were mixed together. Pooling was performed to
enable the characterization of the broad canine uri-
nary proteome across multiple breeds. While this
approach does not allow for observation of individual
differences between dogs, it was chosen to represent
a general population. Batched urine was aliquoted
into 6 ultracentrifuge tubes (25 9 89 mm; Beckman,
Brea, CA, USA), and centrifuged at 17,000g for
10 min at 4°C (L70 Ultracentrifuge, SW 28 rotor;
Beckman) to remove whole cells and debris.5 The
sample supernatants were then collected and aliquot-
ed into 6 clean ultracentrifuge tubes (Beckman).
Finally, the samples were ultracentrifuged at 113,000g
for 2 h at 4°C (L70 Ultracentrifuge, SW 28 rotor; Beck-
man) to obtain exosomal pellets.5 The supernatant
from this ultracentrifugation step was decanted and
stored at �80°C until proteomic analysis of the soluble
fraction of the canine urine could be performed.
The exosomal pellets were resuspended in isolat-
ing buffer (10 mM triethanolamine, 250 mM sucrose
at pH of 7.6; Sigma-Aldrich), and each sample was
transferred to a 1.6 mL Eppendorf tube. Dithiothreitol
(DTT; Sigma-Aldrich) was added to a final concentra-
tion of 200 mg/mL, the entire sample mix was then
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heated at 95°C for 2 minutes for chemical reduction of
Tamm–Horsfall protein (THP).5 Samples were trans-
ferred to 6 ultracentrifuge tubes (14 9 89 mm; Beck-
man). The remaining volume of the ultracentrifuge
tubes was filled with isolation buffer, and the samples
were ultracentrifuged at 200,000g for one h at 4°C(L70 Ultracentrifuge, SW 41 Ti rotor; Beckman).5 The
final pellets were reconstituted in 40 lL of isolation
buffer and combined into 3 1.6 mL Eppendorf tubes,
followed by addition of 5X-SDS Laemmli buffer con-
taining Bromophenol blue and 60 mg/mL DTT at a 1:4
ratio. The sample was incubated at 60°C for 10 min
before boiling for 5 min. All exosomal pellets were
then frozen at�20°C until gel electrophoresis was per-
formed.
One-dimensional SDS-PAGE and in-gel digest ofcanine urinary proteins
Both urine supernatant and exosomal fractions were
thawed on ice. The supernatant was concentrated
using Amicon Ultra-15 centrifugal filter units with a
molecular weight cutoff of 5 kDa (Millipore, Billerica,
MA, USA) as per manufacturer’s instructions. Protein
quantification was performed on both soluble and
Bromophenol blue-dyed free exosomal urine fractions
using the Pierce Coomassie Plus (Bradford) protein
assay kit (Thermo Scientific, Waltham, MA, USA),
according to the manufacturer’s instructions. The con-
centrated supernatant had a protein content of 1.1 lg/lL, and the exosomal pellet contained 1.2 lg/lL of
protein.
Urine supernatant (24.8 lg) and exosomal pro-
teins (19.8 lg) were subjected to electrophoresis in
separate lanes on a 4–20% Mini PROTEAN TGX pre-
cast gel (BioRad, Hercules, CA, USA) with 19 Tris/
Glycine/SDS running buffer, at 175 V for approxi-
mately one hour. The gel was stained with Imperial
Protein Stain (Thermo Scientific, Rockford, IL, USA)
according to manufacturer’s instructions. Prior to
in-gel digestion, each lane was cut into 25 pieces,
which were then diced into ~1 9 1 mm squares and
stored in 0.6 mL Eppendorf tubes.
In-gel digestion was performed using a standard
trypsin digest protocol to produce peptides. Briefly, gel
pieces were destained and washed, after which DTT
reduction and iodacetamide alkylation were per-
formed. Finally, proteins were digested overnight with
mass spectrometry grade trypsin (Promega, Madison,
WI, USA) at 37°C. Tryptic peptides were extracted
from the gel pieces using 50% acetonitrile (ACN) and
0.1% trifluoroacetic acid (Sigma-Aldrich). A vacuum
centrifuge was used to evaporate any remaining sol-
vent from the samples. Samples were reconstituted in
a buffer of 0.1% acetic acid and 3%ACN.
LC-MS/MS analysis
Peptides were purified and concentrated using an
on-line enrichment column (Zorbax C18, 5 lm,
5 9 0.3 mm, Agilent, Santa Clara). Subsequent chro-
matographic separation was performed on a reverse
phase nanospray column (1100 nanoHPLC, Zorbax
C18, 5 lm, 75 lm ID 9 150 mm column; Agilent,
Santa Clara, CA, USA) using a 60-min linear gradient
from 25% to 55% buffer B (90% ACN, 0.1% formic
acid) at a flow rate of 300 nanoliters/min. Peptides
were eluted directly into the mass spectrometer (LTQ
linear ion trap; Thermo Scientific) and spectra were
collected over a m/z range of 200–2000 Da using a
dynamic exclusion limit of 2 MS/MS spectra of a given
peptide mass for 30 s (exclusion duration of 90 s).
Compound lists of the resulting spectra were generated
using Bioworks 3.0 software (Thermo Scientific) with
an intensity threshold of 5000 and one scan/group.
MS/MS spectra were searched against the NCBI
Canine protein sequence database containing 69,862
sequence entries using both the Mascot (version 2.3;
Matrix Science, Boston, MA, USA) and Sequest (Sor-
cerer-SEQUEST; Sage-N Research, Milpitas, CA, USA)
database search engines.
A peptide mass tolerance of 2 Da and a fragment
ion mass tolerance of 1.5 Da were utilized by Mascot
searches, while a peptide mass tolerance of 2 Da and a
fragment ion mass tolerance of 1.0 were used via
Sequest. The following parameters were employed in
all database searches: full tryptic digestion allowing for
one missed cleavage, variable modification of methio-
nine oxidation, and a fixed modification of cysteine
carbamidomethylation.
Peptide identifications from both search engines
were combined using probabilistic protein identifica-
tion algorithms implemented in Scaffold software
(Proteome Software, Portland, OR, USA). Peptide and
protein probability thresholds of 95% and 90%,
respectively, were applied to the results (2.2% FDR as
calculated by Scaffold based on probability statistics).
Proteins containing shared peptides were grouped by
Scaffold to satisfy the laws of parsimony. Manual vali-
dation of MS/MS spectra was performed for all protein
identifications above these thresholds that were based
on one peptide. Selection by manual validation was
based on the following criteria: (1) The peptide must
be identified by both search engines (Sequest andMas-
cot), (2) there must be a minimum of 75% coverage of
theoretical y or b ions (at least 5 in consecutive order),
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(3) there must be an absence of prominent unassigned
peaks greater than 5% of the maximum intensity, and
(4) indicative residue-specific fragmentation, such as
intense ions N-terminal to proline and immediately
C-terminal to aspartate and glutamate were used as
additional parameters of confirmation.
Enrichment analysis of GO categories
Analysis of the canine urinary proteome was modeled
after an earlier report.1 Enrichment analysis of the
total canine urinary proteome dataset was performed
using the Cytoscape plug-in tool, Biological Networks
Gene Ontology (BiNGO).28 Separate datasets were
generated for the full list of exosomal and soluble pro-
teins identified from the canine urine and were indi-
vidually searched against GO annotation terms within
the functional categories. Proteins were appropriately
grouped into GO categories and determined to be over-
or underrepresented in comparison to the complete
International Protein Index (IPI) human proteome
based on results from a hypergeometric test. A resul-
tant P-value of ≤ .001 was considered to be statistically
significant.28 Corrections for multiple term testing
were performed automatically by BiNGO using the
recommended Benjamini and Hochberg correction.28
This analysis was repeated using subset datasets for sol-
uble and exosomal proteins that were common
between dogs and people, as well as soluble and exoso-
mal proteins that were only observed in canine urine.
In all figures representing the data obtained from
BiNGO, GO terms were subjectively paired down to
include as many GO terms encompassing as many
categorized proteins as possible, while still allowing
the data to be presented in a useful manner. Given the
large amount of information, all figures illustrat-
ing the complete BINGO analysis are presented as
supplemental data.
Results
Urine was collected from 15 clinically normal, adult
pet dogs. Four dogs were later excluded due to exces-
sive numbers of acanthocytes onmicroscopic review of
the blood film (2 dogs), mildly elevated ALT activity of
435 IU/L (reference range 10–110 IU/L) and succes-
sive lymphoma (one dog), and marked struvite crys-
talluria on urine sediment examination (one dog).
The remaining 11 dogs had no abnormalities in
CBC, blood film review, serum chemistry profile, or
urinalysis, and had a UPC ≤ 0.1. This group of dogs
included 6 female spayed dogs, one female intact dog,
and 4 male castrated dogs. Dogs ranged from 2 to
9 years of age with a median of 6 years; breeds
included 5 mix-breed dogs, 3 Labrador Retrievers, one
Border Collie, one Golden Retriever, and one Pug.
Characterization of the canine urinary proteome
In total, 563 proteins were confidently identified after
the removal of redundant proteins and anionic tryp-
sinogen from the dataset. Of these, 391 were identified
in the exosome fraction (Table S1) and 214 were iden-
tified in the soluble fraction (Table S2). A total of 349
proteins were detected exclusively in the exosome
fraction (Figure 1), and 172 were detected exclusively
in the soluble fraction, while 42 proteins were identi-
fied in both fractions. A total of 74 proteins in the exo-
some fraction, and 50 proteins in the soluble fraction
were identified based on a single peptide. Manual vali-
dation of the detected spectra for these proteins was
performed as described in the Materials and Methods
section.
Themolecular weight distribution of the identified
proteins in the canine urine was highly similar to that
of people.1,4 The majority of canine urinary proteins,
from both the exosomal and soluble fractions, had
a molecular weight of approximately 10–80 kDa
(Figure 2). Only 64 canine exosomal proteins and 58
soluble proteins had amolecular weight ≥ 80 kDa.
The canine urinary proteome was further charac-
terized using BiNGO, a plug-in for Cytoscape, which
searches proteins against Gene Ontology (GO) terms
and categories.28,29 Gene Ontology was developed by
the GO Consortium in 2000 to unify the annotation of
proteins from various eukaryotic organisms by relying
on a stringently defined vocabulary of terms to be
included in the larger functional categories of Cellular
Component, Molecular Function, and Biological Pro-
cess.29 The ontologic categorization of proteins is fur-
ther enhanced through the GO’s links to various other
gene and protein databases, such as SwissPROT and
Figure 1. Venn diagram illustrating the distribution of total canine
urinary proteins within the exosome and soluble fractions identified by
liquid chromatography tandemmass spectrometry analysis.
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GenBank, which helps the GO protein classifications
to stay current.29
Of the identified proteins, 6 could not be catego-
rized using BiNGO: Chain A, Grp94 N-terminal bound
to geldanamycin (exosome), Na+/K+ ATPase alpha
chain (exosome), IgG heavy chain B (exosome & solu-
ble), IgG heavy chain D (soluble), IgG heavy chain A
(soluble) and DLA class I histocompatibility antigen,
A9/A9 alpha chain (soluble) as standard gene name
abbreviations are not established for these proteins,
which are necessary to input datasets. For the remain-
ing proteins, 2 datasets were established, one com-
prised of 388 exosomal proteins, and the other of 213
soluble proteins, while 42 proteins were similar
between the 2 datasets. Within the GO categories of
Cellular Component, Molecular Function, and Biolog-
ical Process, the number of urinary proteins linked to a
minimum of one annotation term was 335, 331, and
324 from the exosomal fraction, and 190, 175, and 168
from the soluble fraction, respectively. The distribution
of over- and underrepresented proteins within the
respective GO categories representing the entire com-
plement of proteins detected in the canine urine is
shown in Figures S1–S12.
Comparison of canine vs human urinary proteomevia Gene Ontology annotation
Comparison of our canine data with recently published
human data1 revealed that 52% (205/391) of the
canine exosomal proteins and 62% (133/214) of the
canine soluble proteins were also found within the
published human urine proteome as reported in 2006
(Figure 3).1 Interestingly, 48% (186/391) of the exos-
omal and 38% (81/214) of soluble proteins identified
in the present study appear to be unique for dogs.
To further compare the canine urine proteome
with published human data, we subdivided our results
into 2 datasets, the first containing only canine pro-
teins found in common with human urine proteins,
and the second containing proteins that were only
Figure 2. Molecular weight distribution of canine urinary proteins identified in the exosome and soluble fractions by liquid chromatography tandem
mass spectrometry.
Figure 3. Total urinary proteins identified by liquid chromatography
tandem mass spectrometry that are unique to canine urine (gray bars)
vs those common to human urine (black bars) in both the exosome and
soluble fractions.
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detected in our canine study. BiNGO analysis was
repeated with these new datasets, searching for over-
and underrepresented terms within the GO functional
categories of Cellular Component, Molecular Func-
tion, and Biological Process (Figures S13–S30).The distributions of over- and underrepresented
proteins within the GO Cellular Component, Molecu-
lar Function, and Biological Process categories were
highly similar between datasets containing the whole
list of canine proteins and the datasets containing only
proteins common between the canine and human.
Cellular Component (canine/human commonproteins)
The majority of overrepresented proteins within the
Cellular Component group were linked to intracellular
locations (Figure S13–S15). Gene Ontology analysis of
the human urine proteome revealed many soluble
proteins annotated to extracellular terms, with fewer
numbers annotated to intracellular terms.1 Canine
urine was also observed to contain both intracellular
and extracellular components; however, intracellular
proteins were present in greater number in the exoso-
mal fraction, while extracellular proteins were more
common in the soluble fraction.
Molecular Function (canine/human commonproteins)
Many proteins common to both canine and human
urine were annotated to the same Molecular Function
(such as peptidase activity and various peptidase activ-
ity daughter terms) (Figures S16–S19). However, the
relative number of proteins annotated to various GO
terms differed between canine and human urine.1 For
example, canine urine has fewer proteins annotated to
signal transducer activity and enzyme inhibition than
human urine.1 In contrast and similar to human urine,
the majority of underrepresented Molecular Function
terms annotated to canine proteins are associated with
nuclear activities, such as DNA binding.1
Biological Processes (canine/human commonproteins)
The GO Biological Process category resulted in the
largest lists of annotated terms for statistically signifi-
cantly overrepresented proteins in canine urine (Fig-
ure S20 and S21). Again, while the distribution of
terms was similar, the relative number of proteins
annotated to terms such as cell communication,
defense response, and immune response was higher
in human urine.1 Furthermore, only a small number
of canine proteins were annotated to immunologic
terms, such as natural killer cell-mediated immunity,
leukocyte degranulation, and leukocyte-mediated
cytotoxicity.
The primary underrepresented Biological Process
GO terms in the canine dataset represented nuclear
processes, such as regulation of gene expression and
transcription (Figure S22 and S23). Conversely, while
the human dataset contains some proteins annotated
to nucleobase, nucleoside, nucleotide and nucleic acid
metabolism, themajority of underrepresented proteins
in canine urine were annotated to more general terms,
such as cell metabolism and regulation of cellular pro-
cesses.1 This may indicate a prevalence of proteins
related to nuclear biological processes in human as
comparedwith canine urine.
Cellular Component (canine-unique proteins)
As expected, Cellular Component GO terms overrepre-
sented in the exosome fraction primarily annotated to
intracellular locations (Figure S24)1 and soluble pro-
teins primarily annotated to terms related to extracel-
lular components as well as cytoskeletal elements and
components of filtrated plasma (Figure S25). No signif-
icant underrepresented GOCellular Component anno-
tations (Figure S26) were observed for proteins in the
soluble fraction that were only found in canine urine.
This is in contrast to the human data, which contain
many underrepresented proteins annotated to intra-
cellular components, such as organelle, nucleus, and
ribosome.1
Molecular Function (canine-unique proteins)
As expected, the overrepresented Molecular Function
GO terms annotated to exosomal proteins unique to
the dog are few, and include: structural molecule
activity, SNARE (soluble N-ethylmaleimide-sensitive
factor attachment receptor) binding, xylulokinase
activity, and purine-nucleoside phosphorylase activity
(Figure S27 and S28).
The underrepresented exosomal proteins unique
to the dog were annotated to similar Molecular Func-
tion terms as in the total dataset (Figure S29). The
underrepresentation of proteins annotated to signal
and molecular transducer activities suggests possible
decreased transducer activity in canine vs human
urine. No statistically significant underrepresented
terms were observed in the soluble proteins unique
to the dog within the GO Molecular Function
category.
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Biological Process (canine-unique proteins)
No statistically significantly underrepresented exo-
some proteins unique to the dog were annotated to
any GO Biological Process terms. For the soluble pro-
teins unique to the dog, proteins were linked to only 2
GO Biological Process terms, ectoderm development
and epidermis development, each containing the same
annotated proteins (Figure S30). No proteins from the
human data were annotated to either of these GO
Biological Process terms.
Proteins with known disease association
Among our list of identified proteins in canine urine,
60 of these proteins (which are also reported in
human urine) are known to be related to specific ex-
trarenal diseases (Table 1).3 Many of these proteins
were linked to rare human diseases that do not have
a recognized canine counterpart, such as hyperm-
ethioninemia, Bardet-Biedl syndrome, and Papillon-
Lefevre syndrome. However, some proteins linked to
human extrarenal disease do have a canine version
of the disease. For example, many proteins on this
list are related to hypertension, including angio-
tensin 1-converting enzyme (ACE) isoforms 1 and
2, dimethylarginine dimethylaminaophydrolase 1,
glutamyl aminopeptidase, hydroxyprostaglandin
dehydrogenase 15-(NAD), and membrane metallo-
endopeptidase.3 Additionally, urinary proteins asso-
ciated with disorders such as megaloblastic anemia,
cystathioninuria, and Charcot–Marie–Tooth neurop-
athy were identified. Megaloblastic anemia can
result from acquired vitamin B6 deficiency or be
secondary to a genetic mutation; it is reported in
dogs and appears to have a similar pathogenesis as
in people.30 Cystathioninuria has been reported
in Dachshunds and a Scottish Terrier.31 Charcot–
Marie–Tooth-like neuropathies have been diagnosed
clinically in dogs; however, further research on the
pathogenesis is needed to determine if these neuropa-
thies are truly similar to those seen in people.19
Additionally, 14 known and/or potential biomar-
kers for renal disease were detected in our canine sam-
ples (Table 1), including albumin, immunoglobulin-c(IgG), and retinal-binding protein 4 (RBP or RBP4),
clusterin, aquaporin-1, hemopexin, fetuin-A, and
ubiquitin A-52 (UBA52), among others. Furthermore,
proteins known to have altered regulation during renal
disease, localized to the kidney, or involved in genetic
renal diseases were detected and include chloride
intracellular channel 1, solute carrier family 12
(Na+K+Cl� transporters) member 1, and myo-inositol
oxygenase.32–35 Notably, multiple known canine bio-
markers of renal disease, such as C-reactive protein
(CRP), GGT, and N-acetyl-b-glucosaminidase (NAG),
were not identified in this study due to our selection of
clinically normal dogs.36–38
Discussion
Adaptation of protocols developed for human urine
resulted in the identification 563 proteins in pooled
urine from clinically healthy dogs. Dogs are thought to
have more THP in their urine than people. The higher
levels of THP and the incompleteness of the canine
protein database both probably contributed to the
reduced number of proteins identified in our study
compared with recent proteomic studies of human
urine.39 Chemical reduction of THP was performed
using previously described methods; however, these
methods were developed for human urine and are
probably not optimized for the high THP levels found
in canine urine. As carnivores, dogs excrete vitamin A
in their urine, and THP has been identified as the reti-
nol carrier protein in canine urine.39 Development of
additional and/or alternative methods to more effi-
ciently remove THP from canine urine could improve
detection coverage of proteins. Canine urine also con-
tains relatively high levels of albumin. Commercially
available kits have been developed for albumin
removal and could be optimized for their use with
canine urine; however, these methods often result in
the nonspecific depletion of additional proteins of
potential biologic interest.1
The molecular weight distribution of canine uri-
nary proteins was similar to that of human urinary
proteins.1 It is widely accepted that proteins filtered via
the healthy glomerulus are generally < 68 kDa.40 The
majority of canine and human urinary proteins are in
the range of 10–79 kDa, while a smaller number of
proteins with molecular weights ≥ 80 kDa are present
in normal urine from both people and dogs.1 These
higher molecular weight proteins may therefore repre-
sent postglomerular filtration proteins released or
secreted by renal epithelial cells, and an in-depth study
of these proteins could provide valuable information
about real-time renal health.
In regard to cellular components, the highest
number of soluble proteins was allocated to the extra-
cellular region and exosomal proteins to the cytoplasm
and organelles. Plasma membrane also contained
many soluble and exosomal proteins similar to human
urine where all urinary proteins were analyzed
together.1 Our sub datasets of proteins (those in
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Table 1. Canine urinary proteins with known associations with human diseases.
Gene Name Protein Name Related Human Disease
Soluble(S)/
Exosome (EX)
ACE Angiotensin 1-converting enzyme isoform 1 Hypertension2 S & EX
ACE 2 Angiotensin 1-converting enzyme 2 Hypertension2 S & EX
ACY1 Aminoacylase 1 Aminoacylase 1 deficiency EX
AGT Angiotensinogen Hypertension, chronic kidney disease (CKD),
IgA nephropathy11S
AHCY S-adenosylhomocysteine Hypermethioninemia EX
AHSG Fetuin-A Acute renal injury10 S
ALB Albumin precursor Dysalbuminemic hyperthyroxinemia,
hyperthyroxinemia–dysalbuminemic analbuminemia
bisalbuminemia glomerular damage, proximal tubular damage2
S & EX
ALDOA [Fructose-bisphosphate] aldolase A
(aka “lung cancer antigen”)
Aldolase deficiency of red cells; myopathy & hemolytic anemia2 EX
AMN Amnionless protein precursor Megaloblastic anemia 12 EX
APOA 1 Apolipoprotein A-1 precursor Primary hypoalphalipoproteinemia2 EX
AQP 1 Aquaporin 1 AQP1 deficiency, Colton-Null, renal neoplasms, ischemic
reperfusion injury to the kidneys5,23EX
ARL6 ADP-ribosylation factor-(like) 6 Bardet–Biedl syndrome 32 EX
ASL Argininosucinate lyase isoform 3 Argininosuccinic aciduria 2 EX
ASS1 Argininosucinate synthetase 1 Citrullinemia2 EX
ATP6V0A4 ATPase, H+ transporting, lysosomal
V0 subunit a4
Renal tubular acidosis, distal, autosomal recessive2 EX
ATP6V1B1 Atpase, H+ transporting, lysosomal
56/58 kDa, V1 subunit B1
Renal tubular acidosis, distal, w/progressive deafness2 EX
B4GALT1 UDP-Gal:bGlcNac b 1,4-galactosyltransferase
1, membrane-bound form
Congenital disorder of glycosylation type IId2 S
CA2 Carbonic anhydrase II Autosomal recessive syndrome of osteopetrosis with
renal tubular acidosis2EX
CA4 Carbonic anhydrase IV precursor Proximal renal tubular acidosis2 EX
CHMP2B Chromatin modifying protein 2B Frontotemporal dementia, chromosome 3-linked2 EX
CLU Clusterin General renal injury. Glomerular, tubular, and renal
papillary damage. Proximal tubular24S
COL6A3 a 3 type VI collagen (isoform 5 precursor) Ullrich congenital muscular dystrophy2 S & EX
CRYM Crystalline, l isoform (1) Autosomal dominant nonsyndromic deafness2 EX
CTH Cystathionase isoform (2), [we got 1] Cystathioninuria2 EX
CTSA Cathepsin A (precursor) Galactosialidosis2 S
CTSD Cathepsin D Papillon-Lef�evre syndrome2 S & EX
DDAH1 Dimethylarginine dimethylaminohydrolase 1 Hypertension2 EX
DNM2 Dynamin 2 (isoform 4) Charcot–Marie–Tooth neuropathy2 EX
DPYS Dihydropyrimidinase [isoform 1] Dihydropyrimidinuria2 EX
DYSF Dysferlin Miyoshi myopathy2 EX
EFEMP1 EGF-containing fibulin-like extracellular matrix Doyne Honeycomb retinal dystrophy2 S
EGF Epidermal growth factor Acute renal injury39 S & EX
ELA2 Elastase (2), neutrophil Cyclic hematopoiesis2 S
ENPEP Glutamyl aminopeptidase (aminopeptidase A) Hypertension2 S & EX
FBP1 Fructose-1, 6-bisphosphatase 1 Fructose-1, 6-bisphosphatase deficiency2 EX
FGA Fibrinogen, a (polypeptide isoform a-E) Renal amyloidosis, Dysfibrinogenemia2 S
FTL Ferritin, light (polypeptide), [chain] Iron overload, autosomal dominant EX
GAA (Acid) a-glucosidase preprotein Infantile-onset glycogen storage disease Type II2 S
GGT/GGT1 c-glutamyltransferase Proximal tubular damage38,39 EX
GPI Glucose [6-]phosphate isomerase Chronic hemolytic anemia due to GPI deficiency2 EX
GSS Glutathione synthetase Glutathione synthetase deficiency2 EX
HPD 4-Hydroxyphenylpyruvate dioxygenase Tyrosinemia type III2 EX
HPGD Hydroxyprostaglandin dehydrogenase 15-(NAD) Hypertension2 EX
HPX Hemopexin Glomerular disease/damage40 S
(continued)
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common with human and those unique to the canine
urine) followed a similar distribution.
Exosomal proteins yielded significantly lower
numbers of proteins that are integral and intrinsic to
the membrane. These types of proteins are buried
inside, penetrate one side of the plasma membrane’s
lipid bilayer, or span both sides of the lipid bilayer,
respectively. It is somewhat surprising that proteins
embedded in the plasmamembranewould not be pres-
ent in higher numbers in the exosomal fraction. How-
ever, perhaps integral and intrinsic membrane
proteins are preferentially retained by the plasma
membrane during exocytosis of exosomal vesicles.
Nuclear and intracellular proteins were underrepre-
sented in the soluble protein fraction. This is an
expected result as it is unlikely that nuclear and
intracellular proteins would escape from normal,
healthy cells into the renal tubular lumen.
Proteins involved in various Molecular Functions
were found in both the exosomal and soluble urine
fractions. The canine urinary exosomal fraction con-
tains many proteins related to various types of protein
binding and catalase activity. In metabolically active
tissue such as the kidneys, it is not surprising that these
intracellular molecular activities would be amplified in
the exosomal fraction of urine. A similar distribution of
annotated GO Molecular Function terms was found in
the datasets of common proteins and the dataset com-
prised of proteins observed only in canine urine. Pro-
tein binding and hydrolase activity (a GO daughter
term of catalytic activity) are both significantly over-
represented in human urine.1 Proteins involved in
Table 1. (continued).
Gene Name Protein Name Related Human Disease
Soluble(S)/
Exosome (EX)
IgG Immunoglobulin-c Glomerular damage S & EX
KLK1 Kallikrein 1 preprotein Decreased urinary activity of kallikrein2 S
LGALS3 Galectin 3 [binding precursor] Lymphocyte function-associated antigen 12 S & EX
LRRK2 [Ig superfamily containing] Leucine-rich
repeat (kinase 2)
Parkinson’s disease2 S
LYZ Lysozyme precursor Familial visceral amyloidosis, general renal disease6,38 S
MME Membrane metallo-endopeptidase Hypertension2 EX
MYH14 Myosin[-6] (heavy chain 14) isoform 1 Autosomal dominant nonsyndromic sensorineural deafness2 EX
NDRG1 N-myc downstream regulated gene 1 Charcot–Marie–Tooth disease type 4D2 EX
NEB Nebulin [related anchoring protein
isoform S isoform 2]
Nemalin myopathy2 EX
PARK7 DJ-1 protein [isoform 1] Parkinson’s disease2 EX
PHGDH [D-3] Phosphoglycerate dehydrogenase Phosphoglycerate dehydrogenase deficiency2 EX
PKLR Pyruvate kinase (liver, and RBC isoform)
[we got isozymes M1/M2]
Pyruvate kinase deficiency2 EX
PODXL Podocalyxin Glomerular disease, diabetic nephropathy, lupus nephritis,
and IgA nephropathy2EX
PRKCH Protein kinase C (ή) [& caseine kinase
substrate in neurons 3 isoforms]
Cerebral infarction2 EX
PROM1 Prominin 1 [precursor] Autosomal recessive retinal degeneration2 EX
RAB3GAP1 (RAB3)
GTPase-activating protein
Warburg micro syndrome2 EX
RBP4 Retinol-binding protein 4, plasma precursor Retinal-binding protein deficiency, proximal tubular
dysfunction12S
RDX Radixin [moesin-binding phosphoprotein 50] Autosomal recessive deafness2 EX
SCL12A3 Solute carrier family 12, member 3 Gitelman syndrome2 EX
SCL3A1 Solute carrier family 3, member 1 Cystinuria2 EX
TF Transferrin Alzheimer disease2 S
THP Tamm–Horsfall Protein Chronic renal disease, medullary cystic kidney disease,
and familial juvenile hyperuricemic nephropathy16S & EX
TPP1 Tripeptidyl-peptidase 1 Ceroid lipofuscinosis neuronal 22 EX
TSG101 Tumor susceptibility gene Breast Cancer2 EX
UBA52 Ubiquitin A-52 ribosomal protein fusion product 1 Diabetic nephropathy7 EX
UMOD Uromodulin precursor Medullary cystic kidney disease-2, Familial juvenile
hyperuricemic nephropathy2S & EX
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protein binding were also present in large numbers in
the canine urine soluble fraction. Additionally, multi-
ple proteins associated with the binding of other sub-
stances, such as carbohydrates, glycoproteins, lipids,
and vitamins, and involved in enzyme regulation and
inhibition were identified within the canine soluble
fraction.
Proteins that were underrepresented in all canine
datasets were largely related to nuclear activities, such
as nucleic acid binding. This is similar to findings in
human urine, and again, not surprising as nuclear
material and activities should be restricted to the
nucleus in intact, healthy cells.1 In contrast to human
urine, where signal transducer activity is significantly
overrepresented with 275 annotated proteins1, pro-
teins related to signal transducer activity were signifi-
cantly underrepresented in the dataset of unique
soluble canine proteins. Cellular signal transduction,
according to the GO definition, involves conveying a
signal across a cell to trigger a response, such as a
change in cellular function or state. The significance of
this startling difference between the protein composi-
tions of canine vs human urine is not yet understood.
A major GO daughter term of signal transduction is
receptor binding, to which 23 proteins in the canine
urinary soluble fraction are annotated. It may simply
be that fewer, more specialized metabolic functions
occur in canine renal epithelium and glomerular fil-
trate. Further studies are required to fully understand
the basis for this observed difference.
Amongst GO Biological Process terms, canine
urine proteins were heavily annotated to terms related
to metabolic process, response to stimulus, transport,
and anatomical structural development. These terms,
and related terms, were likewise heavily linked to
human urinary proteins. However, human urine also
contained overrepresented proteins annotated to
terms related to the immune response, which was not
observed in the canine data. This may indicate a higher
degree of immunologic activity in normal human
urine vs normal canine urine.
Conclusions
Our proteomic analysis of normal canine urine has
yielded an extensive list of proteins, some of which are
already being used as urinary biomarkers for renal dis-
ease in people. Moreover, proteins such as fetuin-A
and UBA52 have not been previously identified in
canine urine and may prove to be useful indicators of
acute renal injury in dogs. Additionally, proteins asso-
ciated with extrarenal disease, as mentioned above,
were also identified. Thus, the results of this study pro-
vide a framework for future studies of canine renal and
extrarenal diseases, and also suggest that the dog may
represent a viable large animal model to study genetic
diseases, systemic hypertension, neoplasia, and other
human diseases.
Disclosure: The authors have indicated that they have no
affiliations or financial involvement with any organization
or entity with a financial interest in, or in financial competi-
tion with, the subject matter or materials discussed in this
article.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Gene Ontology (GO) Cellular Component
terms significantly overrepresented (P < .001) in the
canine urine exosome fraction in comparison to the
total IPI human proteome. The annotation ratio is the
number of canine exosomal proteins associated with a
specific GO term divided by the total number of canine
exosomal proteins annotated to the specific functional
annotation group by GO. The annotation ratio is calcu-
lated in the samemanner for IPI human proteins and is
defined as the number of IPI proteins associated with a
specific GO term divided by the total number of IPI
human proteins annotated by GO within a functional
group.
Figure S2.GOCellular Component terms significantly
overrepresented (P < .001) in the canine urinary solu-
ble fraction in comparison to the total IPI human pro-
teome.
Figure S3. GO Cellular Component terms significantly
underrepresented (P < .001) in the canine exosomal
fraction in comparison to the total IPI human proteome.
Figure S4.GOCellular Component terms significantly
underrepresented (P < .001) in the canine soluble
fraction in comparison to the total IPI human prote-
ome.
Figure S5. GOMolecular Function terms significantly
overrepresented (P < .001) in canine urinary exosome
fraction in comparison to the total IPI human.
Figure S6. GOMolecular Function terms significantly
overrepresented (P < .001) in canine urinary soluble
fraction in comparison to the total IPI human prote-
ome.
Figure S7. GOMolecular Function terms significantly
underrepresented (P < .001) in the canine exosomal
fraction in comparison to the total IPI human prote-
ome.
Figure S8. GOMolecular Function terms significantly
underrepresented (P < .001) in the canine soluble
fraction in comparison to the total IPI human prote-
ome.
Figure S9. GO Biological Process terms significantly
overrepresented (P < .001) in the canine urinary exo-
some fraction in comparison to the total IPI human
proteome.
Figure S10. GO Biological Process terms significantly
overrepresented (P < .001) in the canine soluble frac-
tion in comparison to the total IPI human proteome.
Figure S11. GO Biological Process terms significantly
underrepresented (P < .001) in the canine exosomal
fraction in comparison to the total IPI human prote-
ome.
Figure S12. GO Biological Process terms significantly
underrepresented (P < .001) in the canine soluble
fraction in comparison to the total IPI human prote-
ome.
Figure S13. Canine/human common exosome pro-
teins. Shown is a selection of major annotated GO Cel-
lular Component terms that are significantly
overrepresented (P < .001) as compared to the total IPI
human proteome. The annotation ratio is calculated as
previously described in Figure S1.
Figure S14. Canine/Human common soluble proteins
overrepresented. A selection of major annotated GO
Cellular Component terms significantly overrepre-
sented (P < .001) as compared to the total IPI human
proteome here.
Figure S15. Canine/Human common soluble proteins
underrepresented. All annotated GO Cellular Compo-
nent terms that are significantly underrepresented
(P < .001) as compared to the total IPI human
proteome.
Figure S16. Canine/Human common exosome pro-
teins. A selection of major annotated GO Cellular
Component terms significantly overrepresented
(P < .001) as compared to the total IPI human prote-
ome.
Figure S17. Canine/Human common soluble proteins.
A selection of major annotated GO Cellular Com-
ponent terms significantly overrepresented (P < .001)
as compared to the total IPI human proteome.
Figure S18. Canine/Human common exosome pro-
teins. All annotated GO Molecular Function terms sig-
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nificantly underrepresented (P < .001) as compared to
the total IPI human proteome.
Figure S19. Canine/Human common soluble pro-
teins. All annotated GO Molecular Function terms sig-
nificantly underrepresented (P < .001) as compared to
the total IPI human proteome.
Figure S20. Canine/Human common exosome pro-
teins. A selection of major annotated GO Biological
Process terms significantly overrepresented (P < .001)
as compared to the total IPI human proteome.
Figure S21. Canine/Human common soluble pro-
teins. A selection of major annotated GO Biological
Process terms significantly overrepresented (P < .001)
as compared to the total IPI human proteome.
Figure S22. Canine/Human common exosome pro-
teins. All annotated GO Biological Process terms signif-
icantly underrepresented (P < .001) as compared to
the total IPI human proteome.
Figure S23. Canine/Human common soluble pro-
teins. All annotated GO Biological Process terms signif-
icantly underrepresented (P < .001) as compared to
the total IPI human proteome.
Figure S24. Canine/Human common soluble pro-
teins. All annotated GO Biological Process terms signif-
icantly underrepresented (P < .001) as compared to
the total IPI human proteome.
Figure S25. Canine unique soluble proteins overrep-
resented. A selection of major annotated GO Cellular
Component terms that are significantly overrepre-
sented (P < .001) as compared to the total IPI human
proteome.
Figure S26. Canine unique exosome proteins under-
represented. All annotated GO Cellular Component
terms that are significantly underrepresented
(P < .001) as compared to the total IPI human prote-
ome.
Figure S27. Canine unique exosome proteins. A selec-
tion of major annotated GO Molecular Function terms
\significantly overrepresented (P < .001) as compared
to the total IPI human proteome.
Figure S28. Canine unique soluble proteins. A selec-
tion of major annotated GO Molecular Function terms
significantly overrepresented (P < .001) as compared
to the total IPI human proteome.
Figure S29. Canine unique exosome proteins. All
annotated GO Molecular Function terms significantly
underrepresented (P < .001) as compared to the total
IPI human proteome.
Figure S30. Canine unique soluble proteins. All anno-
tated GO Biological Process terms that are significantly
overrepresented (P < .001) as compared to the total IPI
human proteome.
Table S1. Proteins identified in the exosome fraction
of canine urine.
Table S2. Proteins identified in the soluble fraction of
canine urine.
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