Urinary proteome of dogs with kidney injury during babesiosis

doi:10.21203/rs.2.11052/v4

Download PDF

Research article

Urinary proteome of dogs with kidney injury during babesiosis

https://doi.org/10.21203/rs.2.11052/v4

This work is licensed under a CC BY 4.0 License

Journal Publication

published 04 Dec, 2019

Read the published version in BMC Veterinary Research →

You are reading this latest preprint version

Background Acute kidney injury is the most frequent complication of babesiosis in dogs and may provide a natural model for identifying early and specific markers of kidney injury in this species. There are limited data on urine proteomics in dogs, and none of the effect of babesiosis on the urine proteome. This study aimed to identify urinary proteins of dogs with kidney injury during the natural course of babesiosis caused by Babesia canis, and to compare them with proteins in a control group to reveal any potential biomarkers predicting renal injury before the presence of azotemia. Urine samples were collected from 10 dogs of various breeds and sex with naturally occurring babesiosis, and 10 healthy dogs. Pooled urine samples from both groups were separated by 2D (two-dimensional) electrophoresis, followed by protein identification using MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometry. Results In total, 176 proteins were identified in the urine samples from healthy dogs, and 403 proteins were identified in the urine samples from dogs with babesiosis. Of the 176 proteins, 146 were assigned exclusively to healthy dogs, and 373 of the 403 proteins were assigned exclusively to dogs with babesiosis; 30 proteins were common for both groups. Characteristic analysis of 373 proteins found in dogs with babesiosis led to the isolation of 8 proteins associated with 10 metabolic pathways involved in immune and inflammatory responses. Conclusions It was hypothesized that epithelial-mesenchymal transition might play an important role in the mechanisms underlying pathological changes in renal tissue during babesiosis, as indicated by a causal relationship network built by combining 5 of the 10 selected metabolic pathways, and 4 of the 8 proteins associated with these pathways; this network included cadherins, gonadotropin releasing hormone receptors, inflammatory responses mediated by chemokine and cytokine signalling pathways, integrins, interleukins, and TGF-β (transforming growth factor β) pathways. Those pathways were linked by interleukin-13, bone morphogenetic protein 7, α2(1) collagen, and tyrosine protein kinase Fer, which are potential biomarkers of damage during babesiosis in dogs, that might indicate early renal injury. Keywords: Acute kidney injury, Babesiosis, Dog, Proteomics, Urine

Small Animal Medicine

Acute kidney injury

Babesiosis

Dog

Proteomics

Urine

After heart failure, kidney disease is the most frequent cause of reduced quality of life and shortened survival of people and dogs [1-4]. Two different forms of kidney disease, acute kidney injury (AKI) and chronic kidney disease (CKD), are caused by various factors. In humans, 7.8% of patients with AKI develop CKD, and 4.9% of patients progress to end-stage renal disease [1]. Most AKI cases in medicine and veterinary science are diagnosed based on serum or plasma concentrations of non-protein nitrogenous creatinine (Cr) and urea compounds. This method has limited sensitivity, and is not suitable for early AKI detection [2],thus it is necessary to identify markers and methods adequate for the early detection of glomeruli, and/or tubule injury before the decreased glomerular filtration rate (GFR) is signalled by increased Crea concentration [3-6]. One such method is proteomic analysis, which compares the protein profile in normal urine with that typical for a given disease to select potential diagnostic, therapeutic, and prognostic biomarkers [7, 8]. With the decreased GFR and subsequent azotemia and uremia, AKI is among the most frequent complications of babesiosis in dogs, and may provide a natural model for identifying early and specific markers of kidney injury in this species [9,10,11,12]. Urinary proteins are a promising target for detecting kidney injury. Only a minimal amount of proteins is present in normal urine, due to the mechanical barrier of the glomerulus, and the reabsorption in the proximal tubules. Urinary total protein (UTP) contains proteins originating from filtered plasma, lower urinary tract, and kidney-derived proteins. High urinary protein concentration can be a result of nephron dysfunction, as healthy glomerular filtration barrier excludes proteins larger than 69 kDa, the molecular weight of albumin. Also it is worth noticing that positively charged proteins pass the glomelural barrier easier than the negatively charged ones. In conditions of disease, glomerular barrier gradually collapses, allowing large amounts of proteins of high, or intermediate weight pass into the ultrafiltrate. Proteins of small molecular weight (>69kDa) are freely filtered in the glomerulus, but are later reabsorbed by the kidney proximal tubules, therefore both primary and secondary tubular dysfunction can result in proteinuria [11].

Possible causes of acute kidney injury in dogs with babesiosis include anaemic hypoxia, hypovolemia, haemoglobinuric nephropathy, and myoglobinuric nephropathy secondary to rhabdomyolysis [13,14,15]. Anoxia, reduced renal blood flow, hypotension, and renal ischaemia probably play more important roles in the development of AKI than haemoglobinuria. Hypoxia results in greater renal tubular injury than haemoglobin, and the nephrotoxic effects of haemoglobin are highly individual [14]. Additionally, as shown by Zygner et al. [16], the increase in serum TNF-α concentration in dogs with canine babesiosis influences the development of hypotension and renal failure.

Moreover, as AKI naturally occurs during babesiosis in dogs, this situation could serve as a good model for select studies on AKI in humans. This hypothesis is supported by comparative analysis of urine proteomes in humans and dogs; many proteins related to human diseases, including kidney diseases, have been identified in canine urine [17-19]. In addition, domestic dogs (Canis lupus familiaris) are increasingly perceived as an excellent animal model for studying complex human diseases [20]. Canine DNA and protein sequences are much closer than mouse sequences to human sequences, suggesting that canine biology is more similar in many aspects to human biology than is mouse biology [21-22]. It is also worth a mention that babesiosis is a zoonotic parasitic infection, and has similar clinical presentation to canine babesiosis [23].

This study aimed to identify proteins in the urine of dogs with subclinical kidney injury during the natural course of babesiosis, and to compare them with proteins in the control group to reveal potential biomarkers predicting renal injury before the presence of azotemia.

Based on the clinicopathological variables, all dogs with babesiosis met the criteria for early phase AKI [24]. They had proteinuria with UPC>0.5, decreased urine specific gravity (average, 1.015) and significantly elevated uIgG/uCr, uTHP/uCr, and uRBP/uCr values, which indicated glomerular and tubular damage.

In this study, 176 proteins were identified in pooled urine samples collected from healthy dogs, and 403 proteins were identified in pooled urine samples collected from dogs with babesiosis. Images of the gels obtained from dogs with babesiosis, and healthy dogs are shown in Figure 1, and Figure 2, respectively. Tables 4 and 5 contain lists of the proteins, along with their names, scores, molecular weights, number of matches, UniProt base accession numbers and hyperlinks (see Appendix: Supplementary Table 4, 5). With Venn diagram software, which shows logical correlations between groups (http://bioinfogp.cnb.csic.es), 146 of the 176 proteins were assigned exclusively to healthy dogs, and 373 of the 403 proteins were exclusively assigned to dogs with babesiosis; 30 proteins were common for both groups. List of 30 common proteins between two groups has been presented in Supplementary data (see Appendix: Supplementary Table 6). From 146 proteins found exclusively in healthy dogs, 128 were identified by Pantherdb software. According to molecular pathways analysis, those were listed in categories as follows: binding, catalytic activity, molecular function regulator, molecular transducer activity, sturctural molecule activity, transcription regulator activity, and transporter activity. Two most prominent molecular functions of those proteins were binding, and catalytic activity, consisting of 40 and 27 proteins, respectively.

To further evaluate the 373 proteins found in only the dogs with babesiosis, the Panther programme (http://www.pantherdb.org) was used to isolate 21 proteins from the Canis familiaris species, which were used to form a collection of potential diagnostic and pathophysiological biomarkers for this disease (Table 1). Further analysis of these 21 proteins led to the isolation of 8 proteins associated with 10 metabolic pathways, that were attributed to immune and inflammatory response development (Table 2). Further analysis indicated that a causal relationship network could be built by combining 5 of the 10 selected metabolic pathways and 4 of the 8 proteins with which the pathways were associated. These pathways included cadherins, gonadotropin releasing hormone receptors, inflammatory responses mediated by chemokine and cytokine signalling pathways, integrins, and TGF-β pathways and were linked by interleukin (IL)-13, bone morphogenetic protein 7, α2(1) collagen, and FER tyrosine kinase.

Table 1. List of Canis familiaris proteins identified in the urine of dogs with babesiosis by MALDI-TOF/TOF.

Nr	Accession^a	Protein name	GO molecular function
1	P06596	Phospholipase A2	phospholipase
2	A4Z944	Zinc finger BED domain-containing protein 5	transcription factor
3	O46392	Collagen alpha-2(I) chain	extracellular matrix structural constituent
4	Q9XSU4	40S ribosomal protein S11	structural constituent of ribosome
5	O97556	Rab GDP dissociation inhibitor beta	G-protein modulator acyltransferase
6	Q9TTY2	Tyrosine-protein kinase Fer	tyrosine kinese activity
7	P19006	Haptoglobin	hemoglobin binding
8	Q75V93	Calcitonin receptor-stimulating peptide 2	peptide hormone
9	Q2KNA0	Cytospin-A	structural component
10	P01321	Insulin	hormone
12	Q9N2I9	Mitochondrial uncoupling protein 3	oxidative phosphorylation
11	E2RK33	Glutamyl-tRNA(Gln) amidotransferase subunit C, mitochondrial	ligase
12	Q32KH5	N-acetylgalactosamine-6-sulfatase	hydrolase
13	P27597	T-cell surface glycoprotein CD3 epsilon chain	transmembrane signalling receptor
14	P24408	Ras-related protein Rab-9A	GTPase
15	P34819	Bone morphogenetic protein 7 (fragment)	growth factor
16	Q5TJE5	Ral guanine nucleotide dissociation stimulator-like 2	guanyl-nucleotide exchange factor
17	Q9N0W9	Interleukin-13	cytokine
18	Q8WMX5	Solute carrier family 15 member 1	transporter
19	O97578	Dipeptidyl peptidase 1 (fragment)	endopeptidase
20	Q861Y6	Nicolin-1	structural component
21	P17716	Islet amyloid polypeptide	hormone

Table 2. List of metabolic pathways and associated urinary proteins in dogs with babesiosis.

No.	Pathway	Protein
1.	CCKR signalling pathway	Calcitonin receptor-stimulating peptide 2
2.	Cadherin signalling pathway	Tyrosine-protein kinase Fer
3.	Gonadotropin-releasing hormone receptor pathway	Insulin/Bone morphogenetic protein 7 (fragment)
4.	Inflammatory response mediated by chemokines and cytokines	Interleukin 13
5.	Insulin/IGF pathway-mitogen activated protein kinase kinase/MAP kinase cascade	Insulin/Insulin-like growth factor
6.	Insulin/IGF pathway-protein kinase B signaling cascade	Insulin/Insulin-like growth factor
7.	Integrin signalling pathway	Collagen alpha-2(I) chain
8.	Ras pathway	Ral guanine nucleotide dissociation stimulator-like 2
9.	T cell activation	T cell surface glycoprotein CD3 epsilon chain
10.	TGF-beta signaling pathway	Bone morphogenetic protein 7 (fragment)

The final bioinformatic analysis of the urine proteome of dogs with babesiosis indicated that at least eight proteins (IL-13, bone morphogenetic protein 7, α2(1) collagen, tyrosine –protein kinase Fer, calcitonin receptor-stimulating peptide 2, insulin/insulin-like growth factor, ral guanine nucleotide dissociation stimulator-like 2 and T cell surface glycoprotein CD3 epsilon chain ) are related to parasitic invasions and renal inflammatory responses. Non-specific immune responses are activated to limit the initial phase of parasitic invasion, or infection by pathogenic micro-organisms. Parasitic invasion initiates type Th2 immune response, characterised by the activation of Th2 lymphocytes, eosinophilia, basophilia, mast cells, and alternatively activated macrophages (AAM). This process is accompanied by the secretion of IgE antibodies and numerous cytokines, such as IL-3, IL-4, IL-5, IL-9, IL-10, IL-13 and TGF-β. It is well known that IL-13 plays a key role in regulating the anti-parasitic response [25] and is a primary factor that induces fibrosis in many chronic contagious and autoimmune diseases [26]. IL-13 increases the concentration of TGF-β, which leads to collagen deposition in lung and kidney tissues [27], by stimulating its production by macrophages via IL-13Rα2 [28, 29]. Fibrosis is considered the final stage in CKD development regardless of the primary cause, and the effector cells of this process include myofibroblasts generated from renal tubule epithelial cells by epithelial-mesenchymal transition (EMT) [30-33]. During this transition, cells lose polarity upon losing certain communication abilities, and degrading the basement membrane. Adhesive molecules that bind both epithelial cells and the basement membrane, such as E-cadherin and integrins, are replaced by mesenchymal cell markers, such as N-cadherin, nonstriated muscle α-actin, vimentin, fibronectin and collagen I. In an early inflammatory environment, EMT maintains renal tissue homoeostasis by inducing structural regeneration and reconstruction after harmful stress. Long-term support of EMT leads to fibrous degeneration as well as structural and functional tissue and organ disorders [34,35]. Pleiotropic TGF-β molecules and BMPs belonging to the transforming growth factor-β superfamily (TGF-βSF) participate in one of the most well-known signalling pathways in EMT [36-38]. Increased TGF-β levels lead to loss of the epithelial phenotype, acquisition of the mesenchymal phenotype and collagen accumulation. Serine-threonine kinase receptors and cytoplasmic proteins (Smads) participate in transmitting TGF-β/BMP pathway signals. Smad3, which is induced by TGF-β stimulation, can bind the Col1A2 gene promoter to activate the expression of type 1α2 collagen, which may accumulate in interstitial tissue and contribute to extracellular matrix (ECM) accumulation, leading to fibrous degeneration of the organ [39]. On the other hand, BMP-7 inhibits fibrosis, exerts anti-inflammatory effects and stimulates the regeneration of damaged kidney tissues [40]. In experimental systems, BMP-7 recombinant protein expression or overexpression inhibits fibrosis in diabetic nephropathy or AKI, TGF-β-initiated EMT and E-cadherin suppression [41]. BMP-7 exerts an anti-inflammatory effect by inhibiting neutrophil, monocyte and macrophage infiltration and activity, as well as by repressing the expression of the proinflammatory cytokines IL-6 and IL-1β, and the proinflammatory chemokines MCP1 and IL-8 [41].

The epsilon chain is one of the four subunits of the CD3 protein complex that combines participation in the activation of T cells after antigen binding. The CD3 epsilon protein is also expressed in proximal and distal tubules, and Henle loops. The presence of this protein in renal tubules results from the participation in the active transport of sodium or hydrogen ions as sodium or proton pumps [42]. It is also believed that CD3 epsilon protein may be involved in communication of signal transduction, similar to T lymphocytes.

Insulin-like growth factor-1 (IGF-1) is produced by the collecting duct of the adult kidney. Its receptors are present in glomeruli, and on the basolateral membrane of renal proximal tubular cells. IGF-1 promotes cell proliferation and inhibits apoptosis by activation of either phosphatidylinositol-3 kinase (PI3K/Akt), or extracellular signal-regulated kinase (ERK)/mitogen-Activated Protein Kinase (MAPK) pathway [43]. Renal IGF-1 has been shown to decrease in course of ischemic injury, and administration of its exogenous form has been reported to accelerate recovery from ischemic acute renal failure. Reduced apoptosis, and enhanced proliferation of tubular epithelial cells were the proposed mechanisms of action responsible for this beneficial effect [44]. The presence of insulin in association with the IGF-I pathway demonstrated in our studies may indicate with some probability, that IGFBP-7 could be a potential biomarker of acute kidney injury in dogs with babesiosis. However, the exact mechanisms underlying this process are not completely understood.

Haptoglobin, and acute phase protein, also appeard among 21 proteins identified in the urine of dogs with babesiosis. Although it was not included in the final network of close connections selected by the Panther system, haptoglobin is considered to be one of the valuable markers used in the assessment of the course of babesiosis in dogs. In the other study, dogs with babesisois had reduced serum level of haptoglobin in relation to normal values [45,46]. In this context, it is worth to ask a question about the correlation between a decreased serum level of haptoglobin and its level in excreted urine.

In this study, protein analysis was performed by mass spectrometry without any pre-treatment of urine. Looking at the gene ontology map derived from the dataset, it appears that urine proteome in dogs with babesiosisis composed of several clusters of proteins. Although our study has a major limitation of using a pooled samples, we beleive that our findings complete in some way the description of urinary signs of a clinical conditions of renal tissue in dogs with babsiosis. Verifying their significance in the diagnosis and prognosis of the disease requires further study.

In summary, to the best of our knowledge, this study is the first to comprehensively analyse the urinary proteome of dogs with babesiosis, demonstrating the association of the identified proteins with this disease, and kidney injury. Urine interleukin-13, bone morphogenetic protein 7, α2(1) collagen and tyrosine-protein kinase Fer are potential biomarkers of kidney injury during babesiosis in dogs that might indicate early renal injury; however, further studies are needed to verify their significance in the diagnosis and prognosis of the disease. Functional analysis of these four proteins indicates that epithelial-mesenchymal transition (EMT) might play an important role in the mechanisms underlying pathological changes in renal tissues during the course of babesiosis.

5.1 Animals and sample collection

Dogs were enrolled during routine admission to Faculty of Veterinary Medicine clinics at the University of Life Sciences in Lublin. Informed consent was obtained from the owners prior to clinical investigations and sample collection. The studies were reviewed and approved by the Ethics Committee of the University of Life Sciences in Lublin (Poland) No 70/2018. All relevant data for inclusion criteria for dogs and values of urine parameters and urinary biomarkers used in the study have already been published [47]. Briefly, the study involved 20 mixed-breed dogs (10 males, 10 females) weighing 5–8 kg (median, 6.2 kg) and aged 2–7 years (median, 4.35 years) that were divided into two groups. All dogs underwent individual clinical and laboratory tests to determine their health status, and to identify signs of kidney damage, particularly in the diseased group. Group 1 (study group, n=10; five males and five females) consisted of dogs naturally infected with B. canis, while group 2 (control group, n=10; five males and five females) consisted of healthy dogs [47]. All dogs in group 1 showed symptoms of babesiosis (apathy, anorexia, changes in urine colour, and pale mucous membranes), and haematology analysis revealed thrombocytopenia (platelets 12–88 × 10⁹/l) and anaemia (erythrocytes 3.5–5.3 × 10¹²/l.) All dogs were nonazotemic, and the serum creatinine concentration remained within the reference range. All dogs in this group had Babesia-positive blood smears, and infection was additionally confirmed by PCR according to the protocol described in other studie [12,16]. Possible co-infections (borreliosis, anaplasmosis, ehrlichiosis) were excluded in all dogs based on PCR and ELISA results [48]. All dogs in group 1 were successfully treated with imidocarb (5 mg/kg s.c.). Dogs in group 2 were clinically healthy and were referred to the clinic for vaccination purposes. Blood smear analysis and PCR for B. canis gave negative results for all animals in group 2. Voided midstream urine samples were collected in the morning before the treatment with imidocarb, and each sample was centrifuged on the day of collection at 500 × g for 10 minutes at 4°C. The supernatants were removed, and protease inhibitors were added (Protease Inhibitor Cocktail, Roche Diagnostic Corp.). Urine protein and Cr concentration were measured by the enzymatic colorimetric method (BS-130 analyser, Mindray), and basic urinalysis with microscopic sediment analysis was performed on fresh urine samples. Urine specific gravity (USG) was measured using a refractometer. The remaining urine was frozen at –80°C for further analysis. Macroscopic evaluation of urine in group 1 showed yellow to dark brown samples, while all group 2 samples were yellow. Urine protein analysis revealed proteinuria in eight of the 10 group 1 dogs, and eight dogs in this group also had a urine protein/creatinine ratio > 0.5. Urine dipstick analysis showed haemoglobinuria in seven of the 10 group 1 dogs, which was severe (+++) in two dogs. Urine specific gravity was decreased in all diseased dogs, with an average value of 1.015 (Table 3). No dogs in the control group had proteinuria or haemoglobinuria. Statistically higher concentrations of urinary biomarkers (uIgG/uCr, uTHP/uCr, and uRBP/uCr) were found in the urine samples from all dogs with babesiosis compared to those from the control animals (p < 0.05), indicating dysfunctional glomerular and tubular kidney regions (Table 3). For proteomic analysis, 10 individual urine samples (0.5 ml each) from groups 1 (affected dogs) and 2 (healthy dogs) were collected and pooled. Pooled sample was made by mixing the same amount of protein of each tear fluid sample. Each pooled urine sample was subjected to desalting on the filter to enable quick ultrafiltration with a high-density coefficient (Amicon Ultra Merck). Protein concentrations were measured with a microlitre spectrophotometer (NANO), and the urine samples were then prepared and subjected to 2D electrophoresis. Each individual gel spot was then analysed by mass spectrometry with the MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) technique.

Table 3. Urinary parameters of renal function and concentration of urinary markers in dogs with babesiosis (group 1) and healthy dogs (group 2) (expressed as the median and range).

Variable	Group 1 (n = 10)	Group 2 (n = 10)	P value
uCr [mg/dl]	58.68 (3.24–120.58)	135.64 (53.91–212.82)	0.02
Specific gravity	1.015 (1.010–1.030)	1.030 (1.015–1.045)	0.02
UPC [mg/mg]	2.3 (0.48–6.24)	0.2 (0.05–0.40)	0.03
uRBP/uCr [mg/g]	24.65 (0.06-76.21)	0.2 (0.09-0.3)	0.03
uTHP/uCr [mg/g]	1.50 (0.13-7.80)	0.2 (0.09-0.3)	0.03
uIgG/uCr[mg/g]	120.78 (0-394.31)	0	0.02

* Measured using the sulfosalicylic acid (SSA) method

UPC = urine protein-to-creatinine ratio, uRBP = urinary retinol binding protein, uTHP = urinary Tamm-Horsfal protein, uIgG = urinary immunoglobulin G, uCrea = urinary creatinine

5.2 2D electrophoresis

Two-dimensional electrophoresis was used to separate the proteins in the tested urine samples [49]. Preliminary tests showed that the optimum amount of protein for 2D electrophoresis is 85 µg; thus, this amount of protein was broken down via a precipitation and purification kit (ReadyPrep™ 2-D Cleanup Kit, Bio-Rad, Warsaw, Poland). The obtained protein pellets were then dissolved in rehydration buffer, and the resulting solutions were applied to a rehydration plate and covered with 17-cm immobilized pH gradient (IPG) linear strips for isoelectric focusing (pH 3-10, Bio-Rad). The gel on the strips was soaked with the protein sample, and the strips were removed after a 12-hour rehydration period and then subjected to electrophoresis in the first dimension (IEF-100 Hoefer; 250 V/30 min; 10 000 V/3 hr; 60 kV/hr, with a current limit of 50 µA/strip hr). Under the influence of the electric field, proteins in the strips migrated to the location corresponding to their isoelectric point. After separation, the IPG strips were prepared for electrophoresis in the second dimension to separate the proteins by molecular mass. Vertical electrophoretic separation was performed in 12.5% polyacrylamide gels with the following current parameters: 600 V/30 mA/100 W in an electrophoretic chamber (PROTEAN® II xi, Bio-Rad). The obtained gels were subjected to a standard colouring procedure with silver in the presence of formaldehyde as a regulator. The protein spots were cut out of the gels, decolourized, reduced and alkylated using dithiothreitol and iodoacetamide [50]. Gel pieces containing proteins were subjected to digestion to obtain shorter peptide fragments. Trypsin digestion occurred in 50 mM ammonium bicarbonate buffer at 37°C for 12 hours (Promega, Trypsin Gold, Mass Spectrometry Grade, Technical Bulletin) [51]. The obtained peptides were subsequently eluted from the gel with a water/acetonitrile/TFA solution (v:v 450:500:50). The extracted peptides were purified using C18 Zip-TIP pipette tips according to the manufacturer's instructions (Merck Chemicals, Billerica, MA, USA, PR 02358, Technical Note) and applied to the MTP AnchorChip 384 plate (Bruker, Bremen, Germany).

5.3 Mass spectrometry

After the protein samples were dried on the MTP AnchorChip 384 plate, the surface was covered with a super-saturated solution of α-cyano-4-hydroxycinnamic acid (HCCA, Bruker), functioning as a matrix mediating energy transmission to the sample. Simultaneously, 0.5 µl of a peptide standard was applied to the calibration fields (Peptide Calibration Standard II, Bruker), which were also covered with the matrix solution. Spectrometric analysis was performed using an UltrafleXtreme III MALDI-TOF/TOF (Bruker), and flexControl 3.3 (Bruker) software was applied for mass spectra collection. The obtained peptides were subjected to mild ionization using the MALDI-TOF instrument in linear mode within the 900-4000 Da mass scope in reflectron mode. The obtained mass spectra were analysed with flexAnalysis 3.4 (Bruker) software as follows: smoothing (Savitsky-Golay method), baseline subtraction (Top Hat baseline algorithm), and peak geometry (Stanford Network Analysis Platform (SNAP) algorithm). All peaks with a signal to noise ratio > 3 qualified for further analysis. Experimental data were analysed using the abovementioned software to exclude peaks originating from trypsin or environmental pollution. To ensure correct identification, the selection of possible post-translational modifications using BioTools 3.2 (Bruker) was essential. Post-translational modifications are derived from both the methodology and metabolic processes in the patients. The obtained spectra were compared to the Swiss-Prot database restricted to “bony vertebrate” taxa using Mascot 2.2 software with a maximum error of 0.3 Da. The results with Mascot scores above 62 were considered statistically significant (p ≤ 0.05). If this threshold was not reached, the fragment ion spectra of chosen peptides were subjected to fragmentation in tandem spectrometry mode [52,53].

5.4 Bioinformatic analysis

Venn diagrams were used to show differences between gene lists of healthy and diseased dogs where the UniProt accession numbers were used.[ 54].

By means of this software it was possible to obtain subset of proteins assigned exclusively to healthy dogs, subset of proteins common to both groups and subset of proteins assigned exlusively to dogs with babesiosis.

Then to study the biological pathway networks and functional classification the UniProt accession numbers of the protein subset assigned to dogs with babesiosis were entered into Panther Classification System [55] . Analysis was carried out selecting Canis lupus familiaris database.

2D: two-dimensional, MALDI-TOF: Matrix-assisted laser desorption ionization time of flight, TGF-β: transforming growth factor, AKI: acute kidney injury, CKD: chronic kidney disease, Cr: creatinine, GFR: glomerular filtration rate, uIgG: urinary immunoglobulin G, uTHP: urinary TammHorsfall Protein, uRBP: urinary retinol-binding protein, Il13: interleukin 13, EMT : epithelial-mesesnchymal transition, ECM: extracellular matrix, IGF-I : insulin-like growth factor, CD3 : cluster of differentiation 3, USG: urine specific gravity, UPC: urine protein to creatinine ratio

Ethics approval and consent to participate

The protocol was approved by the Animal Ethics Board of the Department an Clinic of Animal Internal Diseases, University of Life Sciences in Lublin.

The dog owners were informed about the methods and purpose of the study and gave their written informed consent.

Consent for publication

Not applicable

Availability of data and material

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors have declared that no competing interests exist.

Funding

This work was supported by the Polish National Science Centre (NCN). DW is supported by Preludium grant number UMO-2016/23/N/NZ5/02576. MW is supported by Preludium grant number UMO-2017/25/N/NZ5/01875. The research materials supporting this publication can be accessed by contacting the corresponding authors.

Authors’ contributions

Conceived and designed the experiments: DW, MW, SW. DW, MW contributed to sample collection and laboratory analysis. LA performed PCR analysis. KM performed electrophoresis procedure and mass spectrometry analysis. All authors participated in the interpretataion of results and the preparation of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Dorota Pietras-Ozga, PhD, for technical assistance with the electrophoresis procedure.

Coca SG, Yusuf B, Shlipak MG, Garg AX, Parikh CR. Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis. Am J Kidney Dis. 2009;53:961-73.
Bellomo R, Ronco C, Kellum JA, Mehta RL, Palevsky P. Acute renal failure - definition, outcome measures, animal models, fluid therapy and information technology needs: the Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group. Crit Care. 2004;8:R204-12.
Kellum JA, Devarajan P. What can we expect from biomarkers for acute kidney injury? Biomark Med. 2014;8:1239-45.
Lee YJ, Chang CC, Chan JP, Hsu WL, Lin KW, Wong ML. Prognosis of acute kidney injury in dogs using RIFLE (Risk, Injury, Failure, Loss and End-stage renal failure)-like criteria. Vet Rec. 2011;168:264.
Murray PT, Mehta RL, Shaw A, Ronco C, Endre Z, Kellum JA, et al. Potential use of biomarkers in acute kidney injury: report and summary of recommendations from the 10th Acute Dialysis Quality Initiative consensus conference. Kidney Int. 2014;85:513-21.
Venkataraman R, Kellum JA. Defining acute renal failure: the RIFLE criteria. J Intensive Care Med. 2007;22:187-93.
Gopal J, Muthu M, Chun SC, Wu HF. State-of-the-art nanoplatform-integrated MALDI-MS impacting resolutions in urinary proteomics. Proteomics Clin Appl. 2015;9:469-81.
Thomas S, Hao L, Ricke WA, Li L. Biomarker discovery in mass spectrometry-based urinary proteomics. Proteomics Clin Appl. 2016;10:358-70.
Köster L, Lobetti R, Kelly P. Canine babesiosis: a perspective on clinical complications, biomarkers, and treatment. Vet Med (Auckl). 2015;6:119–128
Mosqueda J, Olvera-Ramírez, Aguilar-Tipacamú AG, et al. Current Advances in Detection and Treatment of Babesiosis. Curr Med Chem. 2012; 19:1504–1518.
De Loor J, Daminet S, Smets P, Maddens B, Meyer E. Urinary Biomarkers for Acute Kidney Injury in Dogs. J Vet Intern Med 2013;27:998–1010.
Adaszek L, Winiarczyk S. Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland. Vet Parasitol. 2008;152:235-41.
Jacobson LS, Lobetti RG. Renal involvement in dogs with babesiosis. 2001. https://www.ingentaconnect.com/content/sabinet/savet/2001/00000072/00000001/art00007. Accessed 4 Apr 2019.
Jacobson LS, Lobetti RG. Rhabdomyolysis as a complication of canine babesiosis. J Small Anim Pract. 1996;37:286-91.
Solano-Gallego L, Sainz A, Roura, X, et al. A review of canine babesiosis: the European perspective. Parasit Vectors. 2016;9:336.
Zygner W, Gójska-Zygner O, Bąska P, Długosz E. Increased concentration of serum TNF alpha and its correlations with arterial blood pressure and indices of renal damage in dogs infected with Babesia canis. Parasitol Res. 2014;113:1499-503.
Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol. 2006;7:R80.
Brandt LE, Ehrhart EJ, Scherman H, Olver CS, Bohn AA, Prenni JE. Characterization of the canine urinary proteome. Vet Clin Pathol. 2014;43:193-205.
Miller I, Presslmayer-Hartler A, Wait R, Hummel K, Sensi C, Eberini I, et al. In between - Proteomics of dog biological fluids. J Proteomics. 2014;106:30-45.
Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, Kamal M, et al. Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature. 2005;438:803-19.
Parker HG, Shearin AL, Ostrander EA. Man's best friend becomes biology's best in show: genome analyses in the domestic dog. Annu Rev Genet. 2010;44:309-36.
Rowell JL, McCarthy DO, Alvarez CE. Dog models of naturally occurring cancer. Trends Mol Med. 2011;17:380-8.
Akel T, Mobarakai N. Hematologic manifestations of babesiosis. Ann Clin Microbiol Antimicrob. 2017;16:6.
Cowgill LD. Iris grading of acute kidney injury: International Renal Interest Society (IRIS), 2012. http://www.iris-kidney.com/guidelines/grading.html.
Tizard I. Veterinary immunology. 9th ed. St. Louis: Elsevier; 2012.
Wynn TA. IL-13 effector functions. Annu Rev Immunol. 2003;21:425-56.
Wynn TA, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med. 2012;18:1028-40.
Brunner SM, Schiechl G, Kesselring R, Martin M, Balam S, Schlitt HJ, et al. IL-13 signaling via IL-13Ralpha2 triggers TGF-beta1-dependent allograft fibrosis. Transplant Res. 2013;2:16.
Fichtner-Feigl S, Strober W, Kawakami K, Puri RK, Kitani A. IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med. 2006;12:99-106.
Benali SL, Lees GE, Castagnaro M, Aresu L. Epithelial mesenchymal transition in the progression of renal disease in dogs. Histol Histopathol. 2014;29:1409-14.
Burns WC, Kantharidis P, Thomas MC. The role of tubular epithelial-mesenchymal transition in progressive kidney disease. Cells Tissues Organs. 2007;185:222-31.
Kuleš J, Mrljak V, Barić Rafaj R, et al. Identification of serum biomarkers in dogs naturally infected with Babesia canis canis using a proteomic approach. BMC Vet Res. 2014;10:111.
Kuleš J, Gotić J, Mrljak V, Barić R. Blood markers of fibrinolysis and endothelial activation in canine babesiosis. BMC Vet Res. 2017;13:82.
Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420-8.
Pieniazek M, Donizy P, Zietek M, Szynglarewicz B, Matkowski R. The role of TGF-beta-related signal transduction pathways in pathogenesis of epithelial-mesenchymal transition as a key element in cancer development and progression. Postepy Hig Med Dosw (Online). 2012;66:583-91.
Bottinger EP, Bitzer M. TGF-beta signaling in renal disease. J Am Soc Nephrol. 2002;13:2600-10.
Fan JM, Ng YY, Hill PA, Nikolic-Paterson DJ, Mu W, Atkins RC, et al. Transforming growth factor-beta regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int. 1999;56:1455-67.
Patel SR, Dressler GR. BMP7 signaling in renal development and disease. Trends Mol Med. 2005;11:512-8.
Meng XM, Chung AC, Lan HY. Role of the TGF-beta/BMP-7/Smad pathways in renal diseases. Clin Sci (Lond). 2013;124:243-54.
Tsujimura T, Idei M, Yoshikawa M, Takase O, Hishikawa K. Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases. World J Stem Cells. 2016;8:288-96.
Gould SE, Day M, Jones SS, Dorai H. BMP-7 regulates chemokine, cytokine, and hemodynamic gene expression in proximal tubule cells. Kidney Int. 2002;61:51-60.
Alroy J, Ucci AA, Azabdoaftari G, Banner BF, Cheville JC. Expression of CD3 antigens in renal tubule epithelium and renal oncocytomas. Pathol Res Pract. 2005;201:803-8.
Tao Y, Pinzi V, Bourhis J, Deutsch E. Mechanisms of disease: signaling of the insulin-like growth factor 1 receptor pathway--therapeutic perspectives in cancer. Nat Clin Pract Oncol. 2007; 4:591-602.
Ding H, Kopple JD, Cohen A, Hirschberg RJ. Recombinant human insulin-like growth factor-I accelerates recovery and reduces catabolism in rats with ischemic acute renal failure Clin Invest. 1993;91:2281-7.
Ulutas B, Bayramli G, Ulutas PA, Karagenc T. Serum concentration of some acute phase proteins in naturally occurring canine babesiosis: a preliminary study. Vet Clin Pathol. 2005;34:144-7.
Matijatko V, Mrljak V, Kis I, et al. Evidence of an acute phase response in dogs naturally infected with Babesia canis. Vet Parasitol. 2007;144:242-50.
Winiarczyk D, Adaszek L, Bartnicki M, Abramowicz B, Lyp P, Madany J, et al. Utility of urinary markers in the assessment of renal dysfunction in canine babesiosis. Tierarztl Prax Ausg K Kleintiere Heimtiere. 2017;45:84-8.
Dziegiel B, Adaszek L, Carbonero A, Lyp P, Winiarczyk M, Debiak P, et al. Detection of canine vector-borne diseases in eastern Poland by ELISA and PCR. Parasitol Res. 2016;115:1039-44.
Miller I. Application of 2D DIGE in animal proteomics. Methods Mol Biol. 2012;854:373-96.
Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis. 1999;20:601-5.
Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996;68:850-8.
Adaszek L, Banach T, Bartnicki M, Winiarczyk D, Lyp P, Winiarczyk S. Application the mass spectrometry MALDI-TOF technique for detection of Babesia canis canis infection in dogs. Parasitol Res. 2014;113:4293-5.
Winiarczyk M, Winiarczyk D, Banach T, Adaszek L, Madany J, Mackiewicz J, et al. Dog Tear Film Proteome In-Depth Analysis. PLoS One. 2015;10:e0144242.
Oliveros, J.C. (2007-2015) Venny. An interactive tool for comparing lists with Venn's diagrams.https://bioinfogp.cnb.csic.es/tools/venny/index.html]
Mi H., Thomas P. (2009) PANTHER Pathway: An Ontology-Based Pathway Database Coupled with Data Analysis Tools. In: Nikolsky Y., Bryant J. (eds) Protein Networks and Pathway Analysis. Methods in Molecular Biology (Methods and Protocols), vol 563. Humana Press.

Table 4. Proteins identified in urine of healthy dogs.
Protein name	Score	Mass	Matches	Access no.	Hyperlink
Endophilin-A2	64	41.7	9	Q2KJA1	http://www.uniprot.org/uniprot/Q2KJA1
BTB/POZ domain-containing protein KCTD1	66	29.7	7	Q719H9	http://www.uniprot.org/uniprot/Q719H9
Prolyl 3-hydroxylase 3	52	82.6	11	Q8IVL6	http://www.uniprot.org/uniprot/Q8IVL6
Essential MCU regulator	54	11.5	4	Q2M2S2	http://www.uniprot.org/uniprot/Q2M2S2
C-X-C motif chemokine 3	43	11.3	4	Q10746	http://www.uniprot.org/uniprot/Q10746
Desmin	65	53.3	10	Q5XFN2	http://www.uniprot.org/uniprot/Q5XFN2
Uromodulin	65	72.9	13	Q862Z3	http://www.uniprot.org/uniprot/Q862Z3
Heat shock factor-binding protein 1	51	8.5	4	O75506	http://www.uniprot.org/uniprot/O75506
Phosphoglucomutase-2	45	69.9	11	Q7TSV4	http://www.uniprot.org/uniprot/Q7TSV4
Methylmalonyl-CoA mutase, mitochondrial	50	83.6	12	Q9GK13	http://www.uniprot.org/uniprot/Q9GK13
Histone H1t	51	22.1	6	P40286	http://www.uniprot.org/uniprot/P40286
General transcription factor II-I	50	110.6	8	A7MB80	http://www.uniprot.org/uniprot/A7MB80
Zinc finger protein 106	68	210.8	19	O88466	http://www.uniprot.org/uniprot/O88466
Protein CutA	76	19.2	6	O60888	http://www.uniprot.org/uniprot/O60888
Protein Lines homolog 1	58	87.5	11	Q8NG48	http://www.uniprot.org/uniprot/Q8NG48
Dihydropyrimidinase-related protein 1	48	62.5	9	Q14194	http://www.uniprot.org/uniprot/Q14194
Interleukin-22	43	20.3	4	Q9GZX6	http://www.uniprot.org/uniprot/Q9GZX6
BTB/POZ domain-containing protein KCTD1	46	29.7	4	Q719H9	http://www.uniprot.org/uniprot/Q719H9
Ribosome-binding protein 1	48	164.8	9	Q28298	http://www.uniprot.org/uniprot/Q28298
Glycogen debranching enzyme	43	176.9	12	Q2PQH8	http://www.uniprot.org/uniprot/Q2PQH8
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3	67	54.2	9	Q28901	http://www.uniprot.org/uniprot/Q28901
60S ribosomal protein L37	58	11.3	8	P79244	http://www.uniprot.org/uniprot/P79244
Interleukin-11	46	21.6	6	P47873	http://www.uniprot.org/uniprot/P47873
Vascular cell adhesion protein 1	63	82.3	13	P19320	http://www.uniprot.org/uniprot/P19320
Ig heavy chain V region AC38 205.12	66	13	4	P06330	http://www.uniprot.org/uniprot/P06330
Collagen alpha-1(XXV) chain	88	65.1	13	Q9BXS0	http://www.uniprot.org/uniprot/Q9BXS0
Sphingosine 1-phosphate receptor 3	51	43	8	Q99500	http://www.uniprot.org/uniprot/Q99500
Vascular cell adhesion protein 1	51	82.3	11	P19320	http://www.uniprot.org/uniprot/P19320
Vacuolar protein sorting-associated protein 4B	41	49.6	6	P46467	http://www.uniprot.org/uniprot/P46467
SPRY domain-containing protein 7	51	22.2	5	Q2T9X3	http://www.uniprot.org/uniprot/Q2T9X3
Myoglobin	48	17.3	7	P02185	http://www.uniprot.org/uniprot/P02185
Retinol-binding protein 2	43	15.8	5	Q08652	http://www.uniprot.org/uniprot/Q08652
Mesenteric estrogen-dependent adipogenesis protein	53	34.6	6	A4IFN2	http://www.uniprot.org/uniprot/A4IFN2
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10, mitochondrial	49	40.9	6	Q0MQB6	http://www.uniprot.org/uniprot/Q0MQB6
Nucleoside diphosphate kinase A	51	17.3	5	Q05982	http://www.uniprot.org/uniprot/Q05982
PR domain zinc finger protein 12	49	40.7	6	A2AJ77	http://www.uniprot.org/uniprot/A2AJ77
Gastric inhibitory polypeptide receptor	53	54	8	P48546	http://www.uniprot.org/uniprot/P48546
Carbohydrate sulfotransferase 1	54	47.5	9	Q9EQC0	http://www.uniprot.org/uniprot/Q9EQC0
Ras-specific guanine nucleotide-releasing factor 1	50	146.3	13	Q13972	http://www.uniprot.org/uniprot/Q13972
Coiled-coil domain-containing protein 184	39	20.7	3	Q52MB2	http://www.uniprot.org/uniprot/Q52MB2
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2	42	10.1	3	Q9NRX3	http://www.uniprot.org/uniprot/Q9NRX3
Elongation factor 1-beta	59	25	7	Q5E983	http://www.uniprot.org/uniprot/Q5E983
Uncharacterized protein C12orf60 homolog	52	28.3	10	Q810N5	http://www.uniprot.org/uniprot/Q810N5
Serum albumin	44	70.6	10	P49822	http://www.uniprot.org/uniprot/P49822
DNA-binding protein RFX5	52	65.7	11	P48382	http://www.uniprot.org/uniprot/P48382
C2 domain-containing protein 3	50	262.6	15	Q4AC94	http://www.uniprot.org/uniprot/Q4AC94
Protein deglycase DJ-1	55	20.1	6	Q95LI9	http://www.uniprot.org/uniprot/Q95LI9
Calmodulin-regulated spectrin-associated protein 1	54	179.9	12	D3Z8E6	http://www.uniprot.org/uniprot/D3Z8E6
Zinc finger protein 101	49	51.9	7	Q8IZC7	http://www.uniprot.org/uniprot/Q8IZC7
Mini-chromosome maintenance complex-binding protein	51	73.8	7	Q9BTE3	http://www.uniprot.org/uniprot/Q9BTE3
Protein SOX-16 (Fragment)	46	6.9	5	Q62247	http://www.uniprot.org/uniprot/Q62247
Essential MCU regulator, mitochondrial	45	11.5	4	Q2M2S2	http://www.uniprot.org/uniprot/Q2M2S2
G protein-coupled receptor kinase 7	52	62.6	7	Q9Z2G7	http://www.uniprot.org/uniprot/Q9Z2G7
Golgi SNAP receptor complex member 1	49	28.6	11	Q2TBU3	http://www.uniprot.org/uniprot/Q2TBU3
Metaxin-2	52	30.1	5	O88441	http://www.uniprot.org/uniprot/O88441
Glutathione S-transferase Mu 1	52	26.1	7	P10649	http://www.uniprot.org/uniprot/P10649
Calpain-2 catalytic subunit	58	80.7	9	Q27971	http://www.uniprot.org/uniprot/Q27971
Pleckstrin homology domain-containing family G member 4B	56	141.6	15	Q96PX9	http://www.uniprot.org/uniprot/Q96PX9
Profilin-4	52	14.6	6	Q9D6I3	http://www.uniprot.org/uniprot/Q9D6I3
EH domain-containing protein 4	52	61.4	12	Q9H223	http://www.uniprot.org/uniprot/Q9H223
Hepcidin	59	9.3	5	Q8MJ80	http://www.uniprot.org/uniprot/Q8MJ80
Glycine receptor subunit beta	52	56.8	7	P48167	http://www.uniprot.org/uniprot/P48167
Sulfotransferase 4A1	51	33.4	8	P63046	http://www.uniprot.org/uniprot/P63046
Unconventional myosin-Id	59	116.9	10	O94832	http://www.uniprot.org/uniprot/O94832
Actin-related protein 2/3 complex subunit 3	48	20.8	6	Q3T035	http://www.uniprot.org/uniprot/Q3T035
Actin-related protein 2/3 complex subunit 3	48	20.8	6	Q3T035	http://www.uniprot.org/uniprot/Q3T035
Autophagy-related protein 16-1	49	68.9	7	Q676U5	http://www.uniprot.org/uniprot/Q676U5
Carbonic anhydrase 5B, mitochondrial	61	36.8	5	Q9Y2D0	http://www.uniprot.org/uniprot/Q9Y2D0
Putative olfactory receptor 2B3	50	36.2	4	O76000	http://www.uniprot.org/uniprot/O76000
Zinc finger protein 75D	51	60.2	10	P51815	http://www.uniprot.org/uniprot/P51815
Trafficking protein particle complex subunit 1	57	16.9	6	Q17QI1	http://www.uniprot.org/uniprot/Q17QI1
Golgi SNAP receptor complex member 1	52	28.6	9	Q2TBU3	http://www.uniprot.org/uniprot/Q2TBU3
Zinc finger protein 491	50	52.9	9	Q8N8L2	http://www.uniprot.org/uniprot/Q8N8L2
Cytoskeleton-associated protein 2-like	61	83.4	12	A5PK21	http://www.uniprot.org/uniprot/A5PK21
Retinoic acid receptor RXR-beta (Fragment)	52	49.8	9	P49743	http://www.uniprot.org/uniprot/P49743
Apolipoprotein A-II	54	11.3	4	E2RAK7	http://www.uniprot.org/uniprot/E2RAK7
Tubulin polymerization-promoting protein family member 2	50	18.5	6	Q4R3A0	http://www.uniprot.org/uniprot/Q4R3A0
Bcl-2-like protein 2	49	20.9	6	Q1RMX3	http://www.uniprot.org/uniprot/Q1RMX3
Mini-chromosome maintenance complex-binding protein	52	73.8	16	A5PJM5	http://www.uniprot.org/uniprot/A5PJM5
Alpha-2,8-sialyltransferase 8F	55	45.4	8	P61647	http://www.uniprot.org/uniprot/P61647
H-2 class I histocompatibility antigen, K-B alpha chain	62	41.7	11	P01901	http://www.uniprot.org/uniprot/P01901
Fibroleukin	53	50.8	9	Q14314	http://www.uniprot.org/uniprot/Q14314
Phosphatidylethanolamine-binding protein 2	52	21.7	6	Q8VIN1	http://www.uniprot.org/uniprot/Q8VIN1
Zinc finger and SCAN domain-containing protein 5A	63	56.9	8	Q9BUG6	http://www.uniprot.org/uniprot/Q9BUG6
Fructose-1,6-bisphosphatase 1	54	37	5	Q3SZB7	http://www.uniprot.org/uniprot/Q3SZB7
Beta-defensin 107A	51	7.9	3	A4H217	http://www.uniprot.org/uniprot/A4H217
Golgi SNAP receptor complex member 1	62	28.6	10	Q62931	http://www.uniprot.org/uniprot/Q62931
Zinc finger protein 624	63	102.5	13	Q9P2J8	http://www.uniprot.org/uniprot/Q9P2J8
Prelamin-A/C	62	74.6	12	P48679	http://www.uniprot.org/uniprot/P48679
Aspartate-tRNA ligase, cytoplasmic	51	57.5	18	P15178	http://www.uniprot.org/uniprot/P15178
Beta-lactoglobulin	69	20.6	6	Q29146	http://www.uniprot.org/uniprot/Q29146
ATP synthase subunit alpha, mitochondrial	63	59.8	16	P25705	http://www.uniprot.org/uniprot/P25705
RUN and FYVE domain-containing protein 2	55	70.8	18	Q8R4C2	http://www.uniprot.org/uniprot/Q8R4C2
Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 2	57	63.5	10	Q3U4I7	http://www.uniprot.org/uniprot/Q3U4I7
Profilin-3	51	15	7	Q8R4C2	http://www.uniprot.org/uniprot/Q9DAD6
Prolyl 3-hydroxylase 3	62	82.6	10	Q8IVL6	http://www.uniprot.org/uniprot/Q8IVL6
Tumor susceptibility gene 101 protein	56	44.1	7	Q99816	http://www.uniprot.org/uniprot/Q99816
Vascular cell adhesion protein 1	51	82.4	10	P29533	http://www.uniprot.org/uniprot/P29533
Ataxin-7	53	93.8	8	Q8R4I1	http://www.uniprot.org/uniprot/Q8R4I1
Gamma-aminobutyric acid receptor subunit alpha-1	53	52.1	6	P08219	http://www.uniprot.org/uniprot/P08219
43 kDa receptor-associated protein of the synapse	63	47.6	10	P12672	http://www.uniprot.org/uniprot/P12672
Protein-arginine deiminase type-2	57	76	9	P20717	http://www.uniprot.org/uniprot/P20717
Heat shock factor-binding protein 1	53	8.6	6	Q9CQZ1	http://www.uniprot.org/uniprot/Q9CQZ1
Non-homologous end-joining factor 1	55	34.1	7	Q6AYI4	http://www.uniprot.org/uniprot/Q6AYI4
Microtubule-associated protein RP/EB family member 1	51	30.1	10	Q5R7Z5	http://www.uniprot.org/uniprot/Q5R7Z5
Protein kish-A	56	8.4	6	Q9CR64	http://www.uniprot.org/uniprot/Q9CR64
Ubiquitin carboxyl-terminal hydrolase 14	56	56.3	9	P40826	http://www.uniprot.org/uniprot/P40826
Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1	64	96.6	12	Q9DBC3	http://www.uniprot.org/uniprot/Q9DBC3
DNA dC->dU-editing enzyme APOBEC-3G	65	45.9	8	Q694B9	http://www.uniprot.org/uniprot/Q694B9
Annexin A10	63	37.8	8	Q9UJ72	http://www.uniprot.org/uniprot/Q9UJ72
Cysteine and glycine-rich protein 2	57	21.8	6	P97314	http://www.uniprot.org/uniprot/P97314
Calmodulin-regulated spectrin-associated protein 1	50	179.9	15	D3Z8E6	http://www.uniprot.org/uniprot/D3Z8E6
Tyrosine-protein phosphatase non-receptor type 12	58	87.2	7	P35831	http://www.uniprot.org/uniprot/P35831
Dual specificity phosphatase DUPD1	55	24.3	6	P0C595	http://www.uniprot.org/uniprot/P0C595
Protein cereblon	53	50.1	10	Q5R6Y2	http://www.uniprot.org/uniprot/Q5R6Y2
Testis-expressed sequence 33 protein	53	30.8	5	O43247	http://www.uniprot.org/uniprot/O43247
Complexin-3	51	17.6	7	Q8WVH0	http://www.uniprot.org/uniprot/Q8WVH0
Plasmalemma vesicle-associated protein	61	50.6	12	Q9WV78	http://www.uniprot.org/uniprot/Q9WV78
Calcium/calmodulin-dependent protein kinase II inhibitor 1	53	8.6	4	A7MBG3	http://www.uniprot.org/uniprot/A7MBG3
BTB/POZ domain-containing protein KCTD1	56	29.7	6	Q719H9	http://www.uniprot.org/uniprot/Q719H9
Threonine synthase-like 2	62	54.8	6	Q86YJ6	http://www.uniprot.org/uniprot/Q86YJ6
Probable tRNA pseudouridine synthase 1	65	36.6	7	Q5M934	http://www.uniprot.org/uniprot/Q5M934
Ras-related protein Rab-7a	51	23.8	8	P51149	http://www.uniprot.org/uniprot/P51149
Essential MCU regulator, mitochondrial	52	11.5	4	Q2M2S2	http://www.uniprot.org/uniprot/Q2M2S2
Golgi SNAP receptor complex member 1	51	28.6	5	O88630	http://www.uniprot.org/uniprot/O88630
Dual specificity phosphatase DUPD1	54	25.5	8	Q68J44	http://www.uniprot.org/uniprot/Q68J44
GTP-binding protein Rheb	53	20.5	5	Q921J2	http://www.uniprot.org/uniprot/Q921J2
Radical S-adenosyl methionine domain-containing protein 2	60	42.4	10	Q9MZU4	http://www.uniprot.org/uniprot/Q9MZU4
Zinc finger protein 624	50	102.5	11	Q9P2J8	http://www.uniprot.org/uniprot/Q9P2J8
Neurofibromin	56	320.5	16	P97526	http://www.uniprot.org/uniprot/P97526
Protein FAM184B	54	121.9	13	Q9ULE4	http://www.uniprot.org/uniprot/Q9ULE4
Phosphomannomutase 2	58	28.4	6	Q3SZJ9	http://www.uniprot.org/uniprot/Q3SZJ9
Isocitrate dehydrogenase [NADP] cytoplasmic	53	47	6	P41562	http://www.uniprot.org/uniprot/P41562
Beta-1,3-galactosyltransferase 4	44	42.9	7	Q5TJE8	http://www.uniprot.org/uniprot/Q5TJE8
Transmembrane protein 238	51	18.1	4	C9JI98	http://www.uniprot.org/uniprot/C9JI98
Protein FAM3C	51	25.0	7	Q810F4	http://www.uniprot.org/uniprot/Q810F4
Coiled-coil domain-containing protein 136	51	133.7	9	Q3TVA9	http://www.uniprot.org/uniprot/Q3TVA9
Protein KHNYN	50	75.1	9	Q80U38	http://www.uniprot.org/uniprot/Q80U38
Retinol-binding protein 2	50	15.8	4	Q08652	http://www.uniprot.org/uniprot/Q08652
Tetratricopeptide repeat protein 36	72	20.7	7	Q3SZV0	http://www.uniprot.org/uniprot/Q3SZV0
E3 ubiquitin-protein ligase RNF152	62	23.1	6	D2H6Z0	http://www.uniprot.org/uniprot/D2H6Z0
Protein RCC2	50	56.8	10	Q9P258	http://www.uniprot.org/uniprot/Q9P258
Signal peptidase complex subunit 2	54	25.3	6	Q5RAY6	http://www.uniprot.org/uniprot/Q5RAY6
Protein myomaker	50	25.1	4	A6NI61	http://www.uniprot.org/uniprot/A6NI61
Apoptosis-enhancing nuclease	57	37.6	9	Q9CZI9	http://www.uniprot.org/uniprot/Q9CZI9
Short-chain specific acyl-CoA dehydrogenase, mitochondrial	53	44.6	9	P16219	http://www.uniprot.org/uniprot/P16219
Fanconi anemia group B protein	61	99.4	12	Q8NB91	http://www.uniprot.org/uniprot/Q8NB91
Dual specificity phosphatase 28	70	18.7	6	Q4G0W2	http://www.uniprot.org/uniprot/Q4G0W2
Succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial	65	50.3	12	Q4R517	http://www.uniprot.org/uniprot/Q4R517
Zinc finger and SCAN domain-containing protein 5A	51	56.9	9	Q9BUG6	http://www.uniprot.org/uniprot/Q9BUG6
Eukaryotic translation initiation factor 4E-binding protein 1	50	12.7	5	Q0P5A7	http://www.uniprot.org/uniprot/Q0P5A7
39S ribosomal protein L30, mitochondrial	57	18.7	6	Q58DV5	http://www.uniprot.org/uniprot/Q58DV5
WAP four-disulfide core domain protein 12	57	12.7	4	A4K2P0	http://www.uniprot.org/uniprot/A4K2P0
UV-stimulated scaffold protein A	55	82.6	12	Q9D479	http://www.uniprot.org/uniprot/Q9D479
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12	52	17.1	4	Q9UI09	http://www.uniprot.org/uniprot/Q9UI09
Tektin-4	59	51.3	8	Q8WW24	http://www.uniprot.org/uniprot/Q8WW24
Heterochromatin protein 1-binding protein 3	65	61.5	12	Q5SSJ5	http://www.uniprot.org/uniprot/Q5SSJ5
Natriuretic peptides B	37	15.1	4	P16859	http://www.uniprot.org/uniprot/P16859
Zinc finger protein 624	59	102.5	12	Q9P2J8	http://www.uniprot.org/uniprot/Q9P2J8
G kinase-anchoring protein 1	55	42.2	5	Q5XIG5	http://www.uniprot.org/uniprot/Q5XIG5
OTU domain-containing protein 6B	59	34	8	Q8N6M0	http://www.uniprot.org/uniprot/Q8N6M0
Tetratricopeptide repeat protein 36	64	20.7	9	Q3SZV0	http://www.uniprot.org/uniprot/Q3SZV0
Leucine-rich repeat and coiled-coil domain-containing protein 1	61	120.6	12	Q69ZB0	http://www.uniprot.org/uniprot/Q69ZB0
Poly [ADP-ribose] polymerase 12	61	80.5	12	Q9H0J9	http://www.uniprot.org/uniprot/Q9H0J9
Cortexin-2	53	9.1	6	P0C2S0	http://www.uniprot.org/uniprot/P0C2S0
Luc7-like protein 3	51	51.9	11	Q3SX41	http://www.uniprot.org/uniprot/Q3SX41
Putative uncharacterized protein encoded by CRHR1-IT1	52	17.2	9	Q96LR1	http://www.uniprot.org/uniprot/Q96LR1
DNA replication licensing factor MCM4	56	97.1	11	P33991	http://www.uniprot.org/uniprot/P33991
Acyl-CoA synthetase family member 2, mitochondrial	63	69	9	Q17QJ1	http://www.uniprot.org/uniprot/Q17QJ1
Transthyretin	45	16.4	3	P49143	http://www.uniprot.org/uniprot/P49143]
Putative uncharacterized protein DKFZp434L187	62	15.1	8	Q9UFV3	http://www.uniprot.org/uniprot/Q9UFV3
Protein AAR2 homolog	52	43.9	10	Q08DJ7	http://www.uniprot.org/uniprot/Q08DJ7
Annexin A5	55	36	5	P08758	http://www.uniprot.org/uniprot/P08758
Apolipoprotein A-II	53	11.3	5	P0DN36	http://www.uniprot.org/uniprot/P0DN36
Retinol-binding protein 4	44	23.4	6	P27485	http://www.uniprot.org/uniprot/P27485
BTB/POZ domain-containing protein KCTD1	49	29.7	6	Q719H9	http://www.uniprot.org/uniprot/Q719H9
Collagen alpha-2(I) chain	45	80.9	11	C0HJP6	http://www.uniprot.org/uniprot/C0HJP6
L-gulonolactone oxidase	55	51	13	Q8HXW0	http://www.uniprot.org/uniprot/Q8HXW0
Zinc finger protein 624	58	102.5	12	Q9P2J8	http://www.uniprot.org/uniprot/Q9P2J8
Cilia- and flagella-associated protein 52	57	69.2	11	Q8N1V2	http://www.uniprot.org/uniprot/Q8N1V2
Autophagy-related protein 16-1	53	68.9	12	Q676U5	http://www.uniprot.org/uniprot/Q676U5
IQ domain-containing protein D	55	51.8	13	Q17QH9	http://www.uniprot.org/uniprot/Q17QH9
Fibroblast growth factor 12	55	27.6	6	P61328	http://www.uniprot.org/uniprot/P61328
Interferon-induced protein with tetratricopeptide repeats 1	52	52.8	12	Q4R5F5	http://www.uniprot.org/uniprot/Q4R5F5
Fanconi anemia group B protein	50	99.4	13	Q8NB91	http://www.uniprot.org/uniprot/Q8NB91
Protein TMEM155	55	14.4	5	Q5R4Y3	http://www.uniprot.org/uniprot/Q5R4Y3
Transmembrane protein 225	58	26.3	5	Q6GV28	http://www.uniprot.org/uniprot/Q6GV28
Telomerase reverse transcriptase	65	128.6	10	O14746	http://www.uniprot.org/uniprot/O14746
Tetratricopeptide repeat protein 36	64	20.7	9	Q3SZV0	http://www.uniprot.org/uniprot/Q3SZV0
Arginine/serine-rich protein 1	52	33.7	8	Q9BUV0	http://www.uniprot.org/uniprot/Q9BUV0
V-set and transmembrane domain-containing protein 2B	61	30.4	6	A6NLU5	http://www.uniprot.org/uniprot/A6NLU5
Ninein	52	245.2	20	Q8N4C6	http://www.uniprot.org/uniprot/Q8N4C6
Electron transfer flavoprotein subunit beta	51	27.9	7	Q68FU3	http://www.uniprot.org/uniprot/Q68FU3
Coiled-coil domain-containing protein 25	53	24.6	6	Q86WR0	http://www.uniprot.org/uniprot/Q86WR0
Protein-arginine deiminase type-2	49	76	8	P20717	http://www.uniprot.org/uniprot/P20717
Radical S-adenosyl methionine domain-containing protein 2	55	42.4	8	Q9MZU4	http://www.uniprot.org/uniprot/Q9MZU4
Golgi SNAP receptor complex member 1	61	28.6	7	O88630	http://www.uniprot.org/uniprot/O88630
BTB/POZ domain-containing protein KCTD1	55	29.7	5	Q719H9	http://www.uniprot.org/uniprot/Q719H9
Zinc finger and SCAN domain-containing protein 5A	57	56.9	9	Q9BUG6	http://www.uniprot.org/uniprot/Q9BUG6
Calcium-binding mitochondrial carrier protein SCaMC-3	51	52.7	9	Q6GQS1	http://www.uniprot.org/uniprot/Q6GQS1

Table 5. Proteins identified in urine of dogs with babesiosis.
Protein name	Score	Mass	Matches	Access no.	Hyperlink
Lipase member N	54	45.7	8	Q5VXI9	http://www.uniprot.org/uniprot/Q5VXI9
Succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial	57	50.3	9	Q4R517	http://www.uniprot.org/uniprot/Q4R517
TATA box-binding protein-like protein 2	51	39.3	5	Q6SJ95	http://www.uniprot.org/uniprot/Q6SJ95
Interferon gamma	53	18.1	5	P01581	http://www.uniprot.org/uniprot/P01581
m7GpppX diphosphatase	57	38.8	9	Q96C86	http://www.uniprot.org/uniprot/Q96C86
Peptidyl-prolyl cis-trans isomerase F, mitochondrial	52	22.	6	P30404	http://www.uniprot.org/uniprot/P30404
Mast cell carboxypeptidase A	52	48.9	6	P15088	http://www.uniprot.org/uniprot/P15088
SprT-like domain-containing protein Spartan	54	56.1	14	G3X912	http://www.uniprot.org/uniprot/G3X912
39S ribosomal protein L50, mitochondrial	54	18.3	9	Q8VDT9	http://www.uniprot.org/uniprot/Q8VDT9
Trafficking protein particle complex subunit 1	55	17	6	Q5NCF2	http://www.uniprot.org/uniprot/Q5NCF2
Desmin	36	53.3	9	Q5XFN2	http://www.uniprot.org/uniprot/Q5XFN2
Dysferlin	57	240	17	Q9ESD7	http://www.uniprot.org/uniprot/Q9ESD7
Leucine-rich repeat-containing protein 14 O	54	55.7	6	A5PJJ5	http://www.uniprot.org/uniprot/A5PJJ5
Coenzyme Q-binding protein COQ10 homolog B, mitochondrial	58	28	10	Q5I0I9	http://www.uniprot.org/uniprot/Q5I0I9
Bactericidal permeability-increasing protein (Fragment)	54	49	6	Q28739	http://www.uniprot.org/uniprot/Q28739
G protein-activated inward rectifier potassium channel 4	51	48.3	6	P48548	http://www.uniprot.org/uniprot/P48548
Gamma-aminobutyric acid receptor subunit beta-2	56	59.3	11	P47870	http://www.uniprot.org/uniprot/P47870
Ankyrin repeat domain-containing protein 42	53	43.6	7	Q8N9B4	http://www.uniprot.org/uniprot/Q8N9B4
Protein FAM83B	62	115.2	9	Q5T0W9	http://www.uniprot.org/uniprot/Q5T0W9
Gamma-aminobutyric acid receptor subunit alpha-4	62	61.3	8	Q9D6F4	http://www.uniprot.org/uniprot/Q9D6F4
Zinc finger protein 624	61	102.5	14	Q9P2J8	http://www.uniprot.org/uniprot/Q9P2J8
Hyaluronan and proteoglycan link protein 2	54	38.6	8	Q9ESM3	http://www.uniprot.org/uniprot/Q9ESM3
HAUS augmin-like complex subunit 2	55	23.4	4	Q5RE16	http://www.uniprot.org/uniprot/Q5RE16
Thromboxane-A synthase	54	60.7	10	P49430	http://www.uniprot.org/uniprot/P49430
Vimentin	54	53.7	12	P20152	http://www.uniprot.org/uniprot/P20152
TD and POZ domain-containing protein 2	51	42.2	10	Q717B2	http://www.uniprot.org/uniprot/Q717B2
Kinase suppressor of Ras 2	62	110	22	Q3UVC0	http://www.uniprot.org/uniprot/Q3UVC0
Cortexin-2	55	9.2	8	Q3URE8	http://www.uniprot.org/uniprot/Q3URE8
Transmembrane protein 240	53	20.3	5	Q5SV17	http://www.uniprot.org/uniprot/Q5SV17
Protein FAM71C	50	27.9	11	Q8NEG0	http://www.uniprot.org/uniprot/Q8NEG0
Carboxylesterase 1E	58	61.8	13	Q64176	http://www.uniprot.org/uniprot/Q64176
Cytochrome P450 3A31	67	58	13	O70537	http://www.uniprot.org/uniprot/O70537
Leucine-rich repeat-containing protein 14	61	55.3	6	Q15048	http://www.uniprot.org/uniprot/Q15048
Protein phosphatase Slingshot homolog 1	66	116.5	15	Q8WYL5	http://www.uniprot.org/uniprot/Q8WYL5
Twinkle protein, mitochondrial	53	77.6	8	Q96RR1	http://www.uniprot.org/uniprot/Q96RR1
Ubiquitin-conjugating enzyme E2 N	53	17.2	4	Q0P5K3	http://www.uniprot.org/uniprot/Q0P5K3
Delta-1-pyrroline-5-carboxylate synthase	55	87.8	7	Q9Z110	http://www.uniprot.org/uniprot/Q9Z110
40S ribosomal protein S11	35	18.6	3	Q9XSU4	http://www.uniprot.org/uniprot/Q9XSU4
Synaptic vesicle membrane protein VAT-1 homolog-like	61	46.2	8	Q9HCJ6	http://www.uniprot.org/uniprot/Q9HCJ6
Glutamyl-tRNA(Gln) amidotransferase subunit C, mitochondrial	40	18	6	E2RK33	http://www.uniprot.org/uniprot/E2RK33
Trafficking protein particle complex subunit 1	63	17	7	Q5NCF2	http://www.uniprot.org/uniprot/Q5NCF2
Rab GDP dissociation inhibitor beta	44	50.8	8	O97556	http://www.uniprot.org/uniprot/O97556
Parvalbumin alpha	54	12.1	10	P20472	http://www.uniprot.org/uniprot/P20472
B-cell lymphoma 6 protein homolog	52	58.8	11	P41183	http://www.uniprot.org/uniprot/P41183
Probable tubulin polyglutamylase TTLL1	53	49.5	9	Q5PPI9	http://www.uniprot.org/uniprot/Q5PPI9
Neurofilament medium polypeptide	50	95.8	7	P12839	http://www.uniprot.org/uniprot/P12839
Suppressor of tumorigenicity 7 protein	55	67.7	9	Q07E08	http://www.uniprot.org/uniprot/Q07E08
Vesicle transport protein USE1	67	30.8	7	Q9CQ56	http://www.uniprot.org/uniprot/Q9CQ56
Protein C12orf4 homolog	67	54.3	8	D4A770	http://www.uniprot.org/uniprot/D4A770
Cell death activator CIDE-A	67	24.8	6	O70302	http://www.uniprot.org/uniprot/O70302
Tryptophan 5-hydroxylase 2	58	56.8	9	Q2HZ26	http://www.uniprot.org/uniprot/Q2HZ26
Kelch-like protein	57	70.2	8	Q2T9Z7	http://www.uniprot.org/uniprot/Q2T9Z7
Ubiquitin carboxyl-terminal hydrolase 37	63	111	12	Q86T82	http://www.uniprot.org/uniprot/Q86T82
Coatomer subunit beta'	60	103.2	6	P35605	http://www.uniprot.org/uniprot/P35605
Centrosomal protein of 170 kDa protein B	61	171.2	11	Q80U49	http://www.uniprot.org/uniprot/Q80U49
Eukaryotic translation initiation factor 4 gamma 2	68	102.6	15	Q62448	http://www.uniprot.org/uniprot/Q62448
Trifunctional enzyme subunit alpha, mitochondrial	51	83.3	6	Q64428	http://www.uniprot.org/uniprot/Q64428
Isovaleryl-CoA dehydrogenase, mitochondrial	55	46.9	6	P12007	http://www.uniprot.org/uniprot/P12007
Pericentrin	60	380.6	32	O95613	http://www.uniprot.org/uniprot/O95613
Hydroxysteroid dehydrogenase-like protein 2	54	45.5	5	A4FUZ6	http://www.uniprot.org/uniprot/A4FUZ6
Ras-related protein Rab-34, isoform NARR	53	21.1	4	P0DI83	http://www.uniprot.org/uniprot/P0DI83
Interferon-induced protein with tetratricopeptide repeats 1	70	55.8	11	Q4R5F5	http://www.uniprot.org/uniprot/Q4R5F5
Cysteine--tRNA ligase, mitochondrial	62	62	12	Q2KIF8	http://www.uniprot.org/uniprot/Q2KIF8
Ubiquitin carboxyl-terminal hydrolase 37	63	111.2	13	F1N5V1	http://www.uniprot.org/uniprot/F1N5V1
Interferon regulatory factor 2-binding protein 1	67	62.6	8	Q8IU81	http://www.uniprot.org/uniprot/Q8IU81
Ubiquitin carboxyl-terminal hydrolase 37	84	111.2	14	F1N5V1	http://www.uniprot.org/uniprot/F1N5V1
Centrosomal protein of 152 kDa	55	197.9	12	O94986	http://www.uniprot.org/uniprot/O94986
E3 SUMO-protein ligase PIAS2	57	64.3	9	Q6AZ28	http://www.uniprot.org/uniprot/Q6AZ28
ATR-interacting protein	61	72.4	11	Q9N077	http://www.uniprot.org/uniprot/Q9N077
T-cell surface glycoprotein CD3 epsilon chain	54	23	4	P27597	http://www.uniprot.org/uniprot/P27597
Rab GDP dissociation inhibitor beta	55	50.8	10	O97556	http://www.uniprot.org/uniprot/O97556
Elongation factor Tu GTP-binding domain-containing protein 1	64	127.1	16	Q8C0D5	http://www.uniprot.org/uniprot/Q8C0D5
HIV Tat-specific factor 1 homolog	57	86.6	12	Q8BGC0	http://www.uniprot.org/uniprot/Q8BGC0
Peptidyl-prolyl cis-trans isomerase A	58	18.1	5	Q9TTC6	http://www.uniprot.org/uniprot/Q9TTC6
Tektin-4	58	51.3	7	Q8WW24	http://www.uniprot.org/uniprot/Q8WW24
Sp110 nuclear body protein	58	79.6	9	Q9HB58	http://www.uniprot.org/uniprot/Q9HB58
Rab11 family-interacting protein 5	57	69.9	10	Q8R361	http://www.uniprot.org/uniprot/Q8R361
Alkyldihydroxyacetonephosphate synthase, peroxisomal	53	73.7	8	O00116	http://www.uniprot.org/uniprot/O00116
Fibroblast growth factor 9	57	23.5	4	P31371	http://www.uniprot.org/uniprot/P31371
Lysozyme C	52	16.9	5	Q659U0	http://www.uniprot.org/uniprot/Q659U0
Phosphoribosyl pyrophosphate synthase-associated protein 2	55	41.2	7	O08618	http://www.uniprot.org/uniprot/O08618
Zona pellucida sperm-binding protein 3	60	47.1	6	P42098	http://www.uniprot.org/uniprot/P42098
Galactoside 2-alpha-L-fucosyltransferase 2	62	39.1	8	O77485	http://www.uniprot.org/uniprot/O77485
39S ribosomal protein L30, mitochondrial	56	18.7	7	Q58DV5	http://www.uniprot.org/uniprot/Q58DV5
Desmin	47	53.3	6	Q5XFN2	http://www.uniprot.org/uniprot/Q5XFN2
Dipeptidyl peptidase 1 (Fragment)	43	50.1	4	O97578	http://www.uniprot.org/uniprot/O97578
Vimentin	50	53.7	8	P20152	http://www.uniprot.org/uniprot/P20152
Serine/threonine-protein kinase 3	53	57.1	9	Q9JI10	http://www.uniprot.org/uniprot/Q9JI10
Neurofilament medium polypeptide	62	95.8	12	P12839	http://www.uniprot.org/uniprot/P12839
Rab GDP dissociation inhibitor beta	62	50.8	8	O97556	http://www.uniprot.org/uniprot/O97556
Zinc finger protein 624	65	102.5	10	Q9P2J8	http://www.uniprot.org/uniprot/Q9P2J8
Uncharacterized protein KIAA1683 homolog	64	87	6	Q8WNU4	http://www.uniprot.org/uniprot/Q8WNU4
Phosphatidylserine decarboxylase proenzyme	56	47.7	5	Q58DH2	http://www.uniprot.org/uniprot/Q58DH2
Probable tubulin polyglutamylase TTLL1	66	49.4	9	Q0VC71	http://www.uniprot.org/uniprot/Q0VC71
ATP synthase subunit d, mitochondrial	63	18.7	5	P13620	http://www.uniprot.org/uniprot/P13620
Breast cancer anti-estrogen resistance protein 3	61	93.5	8	Q58DL5	http://www.uniprot.org/uniprot/Q58DL5
Sperm surface protein Sp17	61	17.4	4	Q15506	http://www.uniprot.org/uniprot/Q15506
Actin-related protein T1	55	42.1	6	Q4R821	http://www.uniprot.org/uniprot/Q4R821
Inhibitor of nuclear factor kappa-B kinase subunit beta	54	87.8	7	O88351	http://www.uniprot.org/uniprot/O88351
Glucosamine-6-phosphate isomerase 2	53	31.3	5	Q9CRC9	http://www.uniprot.org/uniprot/Q9CRC9
E3 ubiquitin-protein ligase TRIM32	55	73.5	9	Q13049	http://www.uniprot.org/uniprot/Q13049
Protein FAM98B	56	45.9	6	Q80VD1	http://www.uniprot.org/uniprot/Q80VD1
DNA topoisomerase 2-alpha	56	173.5	11	Q01320	http://www.uniprot.org/uniprot/Q01320
Interferon alpha-1/13	50	22.1	4	P01562	http://www.uniprot.org/uniprot/P01562
Bone morphogenetic protein 7	56	21.5	6	P34819	http://www.uniprot.org/uniprot/P34819
Protein phosphatase Slingshot homolog 1	60	116.5	16	Q8WYL5	http://www.uniprot.org/uniprot/Q8WYL5
Serum albumin	42	70.6	14	P49822	http://www.uniprot.org/uniprot/P49822
Peroxiredoxin-1	61	22.3	9	Q6B4U9	http://www.uniprot.org/uniprot/Q6B4U9
Zinc finger BED domain-containing protein 5	48	80.2	11	A4Z944	http://www.uniprot.org/uniprot/A4Z944
Zinc finger and SCAN domain-containing protein 9	57	47	9	O15535	http://www.uniprot.org/uniprot/O15535
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2	69	11.1	7	Q4R5E2	http://www.uniprot.org/uniprot/Q4R5E2
Tektin-4	67	51.3	11	Q8WW24	http://www.uniprot.org/uniprot/Q8WW24
Elongation factor Tu GTP-binding domain-containing protein 1	70	127.1	16	Q8C0D5	http://www.uniprot.org/uniprot/Q8C0D5
DCC-interacting protein 13-beta	68	75	7	Q8NEU8	http://www.uniprot.org/uniprot/Q8NEU8
Ras-related protein Rab-28	55	25	5	Q3SWY9	http://www.uniprot.org/uniprot/Q3SWY9
Translation initiation factor eIF-2B subunit delta	55	58.4	8	Q63186	http://www.uniprot.org/uniprot/Q63186
D-beta-hydroxybutyrate dehydrogenase, mitochondrial	62	15.2	7	P86198	http://www.uniprot.org/uniprot/P86198
Gap junction alpha-8 protein	69	49.9	12	Q8K4Q9	http://www.uniprot.org/uniprot/Q8K4Q9
Dual specificity phosphatase 28	54	18.7	7	Q4G0W2	http://www.uniprot.org/uniprot/Q4G0W2
39S ribosomal protein L30, mitochondrial	53	18.7	7	Q58DV5	http://www.uniprot.org/uniprot/Q58DV5
Ras-related protein Rab-36	61	36.8	12	O95755	http://www.uniprot.org/uniprot/O95755
tRNA-dihydrouridine(20) synthase [NAD(P)+]-like	50	55.8	10	Q9NX74	http://www.uniprot.org/uniprot/Q9NX74
AP-3 complex subunit mu-2	55	47.2	5	P53677	http://www.uniprot.org/uniprot/P53677
OTU domain-containing protein 6B	52	34	7	Q8N6M0	http://www.uniprot.org/uniprot/Q8N6M0
A-kinase anchor protein 10, mitochondrial	57	74.1	10	O88845	http://www.uniprot.org/uniprot/O88845
Hemoglobin subunit beta	56	16.3	6	P02073	http://www.uniprot.org/uniprot/P02073
Single-pass membrane and coiled-coil domain-containing protein 2	55	34.1	8	Q95JR4	http://www.uniprot.org/uniprot/Q95JR4
Myotrophin	56	13.1	5	Q3T0F7	http://www.uniprot.org/uniprot/Q3T0F7
Zinc finger protein 622	77	54.8	17	Q969S3	http://www.uniprot.org/uniprot/Q969S3
Protein POF1B	63	68.9	10	Q8WVV4	http://www.uniprot.org/uniprot/Q8WVV4
HORMA domain-containing protein 1	57	45.4	8	D3ZWE7	http://www.uniprot.org/uniprot/D3ZWE7
Sperm surface protein Sp17	58	17.4	7	Q15506	http://www.uniprot.org/uniprot/Q15506
Tyrosine-protein kinase BAZ1B	56	172.2	14	Q9Z277	http://www.uniprot.org/uniprot/Q9Z277
Nicolin-1	43	24.6	8	Q861Y6	http://www.uniprot.org/uniprot/Q861Y6
Parvalbumin alpha	63	12.1	11	P20472	http://www.uniprot.org/uniprot/P20472
Vimentin (Fragment)	58	51.9	12	P48670	http://www.uniprot.org/uniprot/P48670
Protein FAM3C	52	24.9	7	Q92520	http://www.uniprot.org/uniprot/Q92520
Uncharacterized aarF domain-containing protein kinase 5	53	66.3	8	Q3MIX3	http://www.uniprot.org/uniprot/Q3MIX3
Interferon-induced protein with tetratricopeptide repeats 1	51	55.8	9	P09914	http://www.uniprot.org/uniprot/P09914
Oxidoreductase HTATIP2	50	27.4	10	A2T7G9	http://www.uniprot.org/uniprot/A2T7G9
Glutathione S-transferase alpha M14	52	25.4	10	GSTA1_PIG	http://www.uniprot.org/uniprot/P51781
Sperm acrosome membrane-associated protein 3	50	18.8	8	B6VH75	http://www.uniprot.org/uniprot/B6VH75
Protein FAM204A	53	27.1	6	Q8C6C7	http://www.uniprot.org/uniprot/Q8C6C7
Ras-related protein Rab-36	55	36.8	8	O95755	http://www.uniprot.org/uniprot/O95755
Neurofibromin	50	323.1	24	Q04690	http://www.uniprot.org/uniprot/Q04690
Oxidoreductase HTATIP2	52	27.4	6	A2T7G9	http://www.uniprot.org/uniprot/A2T7G9
Transmembrane emp24 domain-containing protein 9	61	27.5	6	Q3T133	http://www.uniprot.org/uniprot/Q3T133
Centromere protein H	61	28	8	Q3T0L1	http://www.uniprot.org/uniprot/Q3T0L1
Centrosomal protein of 104 kDa	72	105	14	Q80V31	http://www.uniprot.org/uniprot/Q80V31
Nicolin-1	55	24.5	6	Q9BSH3	http://www.uniprot.org/uniprot/Q9BSH3
Acylphosphatase-2	57	10.9	5	P35745	http://www.uniprot.org/uniprot/P35745
Mitofusin-1	58	84.5	9	Q811U4	http://www.uniprot.org/uniprot/Q811U4
Ras-related protein Rab-36	54	36.8	9	O95755	http://www.uniprot.org/uniprot/O95755
Hippocalcin-like protein 1	51	22.4	7	P62748	http://www.uniprot.org/uniprot/P62748
Stanniocalcin-2	47	34.1	9	Q5RAT2	http://www.uniprot.org/uniprot/Q5RAT2
Major vault protein	51	96.2	14	Q9EQK5	http://www.uniprot.org/uniprot/Q9EQK5
Peptidyl-prolyl cis-trans isomerase FKBP1A	59	12	4	Q62658	http://www.uniprot.org/uniprot/Q62658
Tyrosine-protein phosphatase non-receptor type 12	54	87.2	17	P35831	http://www.uniprot.org/uniprot/P35831
Kinase suppressor of Ras 2	52	108.9	16	Q6VAB6	http://www.uniprot.org/uniprot/Q6VAB6
Putative ATP-dependent RNA helicase DHX30	54	136.9	10	Q2NKY8	http://www.uniprot.org/uniprot/Q2NKY8
U3 small nucleolar RNA-associated protein 14 homolog A	53	88.2	10	Q3T0Q8	http://www.uniprot.org/uniprot/Q3T0Q8
39S ribosomal protein L30, mitochondrial	64	18.7	7	Q58DV5	http://www.uniprot.org/uniprot/Q58DV5
Growth arrest and DNA damage-inducible proteins-interacting protein 1	63	25.9	8	Q9CR59	http://www.uniprot.org/uniprot/Q9CR59
Protein FAM57B	66	31.2	7	Q71RH2	http://www.uniprot.org/uniprot/Q71RH2
Ras-related protein Rab-9A	35	23.1	4	P24408	http://www.uniprot.org/uniprot/P24408
Probable tubulin polyglutamylase TTLL1	53	49.5	6	Q5PPI9	http://www.uniprot.org/uniprot/Q5PPI9
Adenylyl cyclase-associated protein 2	51	53.1	8	Q9CYT6	http://www.uniprot.org/uniprot/Q9CYT6
Collagen alpha-1(I) chain (Fragments)	55	75.2	12	C0HJP1	http://www.uniprot.org/uniprot/C0HJP1
NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial	54	51.4	8	Q0MQI4	http://www.uniprot.org/uniprot/Q0MQI4
Rho GTPase-activating protein 18	54	75.2	9	Q8N392	http://www.uniprot.org/uniprot/Q8N392
Rho GTPase-activating protein 39	65	122.2	11	Q9C0H5	http://www.uniprot.org/uniprot/Q9C0H5
Unconventional myosin-Ie	71	127.4	13	Q63356	http://www.uniprot.org/uniprot/Q63356
Zinc finger and SCAN domain-containing protein 25	55	62.6	8	Q6NSZ9	http://www.uniprot.org/uniprot/Q6NSZ9
Four and a half LIM domains protein 2	55	34.1	5	O35115	http://www.uniprot.org/uniprot/O35115
Putative uncharacterized protein MYH16	59	128,4	16	Q9H6N6	http://www.uniprot.org/uniprot/Q9H6N6
EGF domain-specific O-linked N-acetylglucosamine transferase	61	62.6	12	A0JND3	http://www.uniprot.org/uniprot/A0JND3
Annexin A10	55	37.8	6	Q9UJ72	http://www.uniprot.org/uniprot/Q9UJ72
Beta-crystallin B2	51	37.8	4	P02522	http://www.uniprot.org/uniprot/P02522
Tripartite motif-containing protein 42	51	85.7	8	Q8IWZ5	http://www.uniprot.org/uniprot/Q8IWZ5
Syndecan-4	53	21.5	4	O35988	http://www.uniprot.org/uniprot/O35988
OTU domain-containing protein 6B	53	34	7	Q8N6M0	http://www.uniprot.org/uniprot/Q8N6M0
Desmin	65	53.6	7	O62654	http://www.uniprot.org/uniprot/O62654
Cytospin-A	50	125	10	Q2KNA0	http://www.uniprot.org/uniprot/Q2KNA0
OTU domain-containing protein 6B	53	34	7	Q8N6M0	http://www.uniprot.org/uniprot/Q8N6M0
Glutathione S-transferase alpha M14	66	25.4	6	P51781	http://www.uniprot.org/uniprot/P51781
Ribosome-binding protein 1	53	164.8	13	Q28298	http://www.uniprot.org/uniprot/Q28298
Zinc finger protein castor homolog 1	56	193.3	18	Q86V15	http://www.uniprot.org/uniprot/Q86V15
Sorting nexin-3	53	18.8	6	Q1RMH8	http://www.uniprot.org/uniprot/Q1RMH8
Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial	61	32.6	6	Q9CQA3	http://www.uniprot.org/uniprot/Q9CQA3
Coiled-coil domain-containing protein 122	60	32.4	7	Q5T0U0	http://www.uniprot.org/uniprot/Q5T0U0
Zinc finger C2HC domain-containing protein 1C	58	61.1	15	Q9BGW4	http://www.uniprot.org/uniprot/Q9BGW4
Ceramide synthase 3	58	61.1	8	Q8IU89	http://www.uniprot.org/uniprot/Q8IU89
Tetratricopeptide repeat protein 36	55	20.7	8	Q3SZV0	http://www.uniprot.org/uniprot/Q3SZV0
Ras-related protein Rab-17	51	23.7	9	Q9H0T7	http://www.uniprot.org/uniprot/Q9H0T7
Transmembrane and coiled-coil domain-containing protein 5A	52	34.5	5	Q8N6Q1	http://www.uniprot.org/uniprot/Q8N6Q1
Inosine triphosphate pyrophosphatase	56	21.8	6	Q9BY32	http://www.uniprot.org/uniprot/Q9BY32
Vacuolar protein sorting-associated protein 29	51	20.7	6	Q9UBQ0	http://www.uniprot.org/uniprot/Q9UBQ0
AP-3 complex subunit mu-2	66	47.2	8	P53677	http://www.uniprot.org/uniprot/P53677
Ras and EF-hand domain-containing protein homolog	60	71.3	10	Q5RI75	http://www.uniprot.org/uniprot/Q5RI75
Protein C19orf12 homolog	65	15.1	6	Q8WUR0	http://www.uniprot.org/uniprot/Q8WUR0
Serine palmitoyltransferase 2	54	63.6	9	O15270	http://www.uniprot.org/uniprot/O15270
Thioredoxin, mitochondrial	53	18.4	4	P97493	http://www.uniprot.org/uniprot/P97493
Vesicle transport protein USE1	57	30.8	9	Q9CQ56	http://www.uniprot.org/uniprot/Q9CQ56
Far upstream element-binding protein 3 O	55	61.9	7	Q96I24	http://www.uniprot.org/uniprot/Q96I24
Nesprin-3	51	112.3	20	Q4FZC9	http://www.uniprot.org/uniprot/Q4FZC9
Zinc finger protein 532	50	112.2	15	Q6NXK2	http://www.uniprot.org/uniprot/Q6NXK2
Hemoglobin subunit epsilon (Fragment)	50	15.6	6	O13071	http://www.uniprot.org/uniprot/O13071
Vimentin	60	53.7	15	P20152	http://www.uniprot.org/uniprot/P20152
Centromere protein H	61	28	7	Q3T0L1	http://www.uniprot.org/uniprot/Q3T0L1
Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 1	60	93.4	11	Q9MZS1	http://www.uniprot.org/uniprot/Q9MZS1
Pleckstrin homology-like domain family A member 3	59	13.9	5	Q9WV95	http://www.uniprot.org/uniprot/Q9WV95
Zinc finger and SCAN domain-containing protein 5A	71	56.9	11	Q9BUG6	http://www.uniprot.org/uniprot/Q9BUG6
Signal recognition particle receptor subunit beta	55	29.7	7	Q4FZX7	http://www.uniprot.org/uniprot/Q4FZX7
Tetratricopeptide repeat protein 6	53	60	8	Q86TZ1	http://www.uniprot.org/uniprot/Q86TZ1
Protein phosphatase 1 regulatory subunit 36	53	47.7	8	D3Z0R2	http://www.uniprot.org/uniprot/D3Z0R2
Oculomedin	53	5.3	4	Q9Y5M6	http://www.uniprot.org/uniprot/Q9Y5M6
Interleukin-2	57	17.8	6	Q95KP3	http://www.uniprot.org/uniprot/Q95KP3
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2	54	11	5	O43678	http://www.uniprot.org/uniprot/O43678
Ras-related protein Rab-36	57

Urinary proteome of dogs with kidney injury during babesiosis

Status:

Journal Publication

Version 4

Abstract

Figures

Background

Results

Discussion

Conclusions

Methods

Abbreviations

Declarations

References

Supplementary Tables