Skip to main content
Download PDF

Serum and urinary monocyte chemoattractant protein-1 as markers of inflammation and renal damage in dogs with naturally occurring leishmaniosis

Parasites & Vectors volume 17, Article number: 366 (2024) Cite this article

Abstract

Background

Renal disease in canine leishmaniosis is of great importance owing to increased risk of mortality. In human visceral leishmaniosis, monocyte chemoattractant protein-1 (MCP-1) has been used as a marker of renal damage and inflammation. The purpose of this study was first to determine the serum MCP-1 and urinary MCP-1-to-creatinine ratio (uMCP-1/Cr) in healthy dogs and dogs with leishmaniosis at diagnosis, and second to determine whether these markers can differentiate disease severity at diagnosis.

Methods

In total, 19 healthy seronegative dogs and 38 dogs with leishmaniosis were included in the study. Dogs with leishmaniosis were classified as LeishVet clinical staging and as International Renal Interest Society (IRIS) staging. Serum and urinary MCP-1 concentrations were measured with an enzyme-linked immunosorbent assay. A receiver operating characteristic (ROC) curve determined disease severity at diagnosis between two LeishVet groups (Stage II versus stage III and IV).

Results

Dogs in Leishvet stages IIb, III, and IV had a median serum MCP-1 and uMCP-1/Cr concentration higher than healthy dogs (P < 0.0001). No statistical differences were found in serum MCP-1 and uMCP-1/Cr between dogs in LeishVet stage IIa and healthy dogs. The dogs in LeishVet stage IV had significantly higher serum MCP-1 and uMCP-1/Cr compared with the dogs in LeishVet stage IIa (P < 0.0001). Serum MCP-1 and uMCP-1 were significantly higher in dogs in IRIS stage I and II + III + IV compared with healthy dogs. Dogs stage II + III + IV of IRIS had a significantly higher serum MCP-1 compared with dogs in IRIS stage I (P < 0.0001). The area under the ROC curve for serum MCP-1 was 0.78 [95% confidence interval (CI) 0.64–0.93] and for uMCP-1/Cr it was 0.86 (95% CI, 0.74–0.99). The optimal cutoff value for serum MCP-1 and uMCP-1/Cr was 336.85 pg/ml (sensitivity of 79% and specificity of 68%) and 6.89 × 10−7 (sensitivity of 84% and specificity of 79%), respectively.

Conclusions

Serum MCP-1 and uMCP-1/Cr are increased in dogs with leishmaniosis compared with healthy dogs, suggesting the presence of inflammation and renal injury. Serum MCP-1 and uMCP-1/Cr were more elevated in the advanced stages of the disease compared with the moderate stages and, therefore, can be markers of the severity of the disease process.

Graphical Abstract

Background

Leishmania infantum is a protozoan parasite that can cause a wide spectrum of clinical manifestations in infected dogs, with skin lesions being the most frequent clinical manifestation among them and other general clinical signs depending on the organs involved [1]. Following transmission, parasites multiply in the skin at the infection site, can migrate into the viscera, and if they colonize the kidneys, or if the circulating immune complexes that formed are deposited in the kidneys, renal disease can develop and the eventual progression to chronic kidney disease may be fatal for the dog since chronic kidney disease is considered the main cause of death in dogs with leishmaniosis [2, 3].

Leishmania infantum infection in dogs can cause inflammation that varies according to the severity of the disease (based on the different clinical and clinicopathological findings) as shown by several studies on naturally occurring and experimental canine leishmaniosis (CanL) that demonstrated an increase in positive acute phase proteins, such as haptoglobin, C-reactive protein, ceruloplasmin, serum amyloid A, and ferritin, or a decrease in negative acute phase proteins, such as paraoxonase-1 and apoliprotein-A1, during various stages of the disease [4,5,6,7]. Acute phase proteins can change in concentration when systemic inflammation occurs [8] and in a variety of different infectious, inflammatory, and neoplastic diseases [9, 10]. Ideally, owing to the increased mortality risk of dogs with kidney disease in leishmaniosis, it would be desirable to have a biomarker capable of showing renal inflammation and damage before the development of established renal disease that can lead to a reduction in renal function over time.

Crucial for defense against L. infantum infection is the host’s ability to mount a cell-mediated immune response capable of controlling and/or eliminating the parasite [11]. During this process, various chemokines play a fundamental role in attracting specific leucocytes, participating in cell-mediated-immunity, cell activation, and antileishmanial activity [12, 13]. In human leishmaniasis, cytokine / chemokine concentrations are modulated differently depending on the clinical forms of the disease and the causative species of Leishmania [14]. In dogs, immune control of L. infantum requires a balance between proinflammatory T helper 1 type CD4 + cells to control parasite replication and T regulatory 1 cells that mediate an immunosuppressive regulatory response that leads to leishmaniosis progression [15].

Monocytes chemoattractant protein-1 (MCP-1) is a member of the C–C family of chemokines that mobilizes monocytes from the bone marrow to the site of inflammation. Many types of cells, such as monocytes, fibroblasts, astrocytes, mast cells, and endothelial cells, produce MCP-1 that responds to inflammation [16,17,18,19]. Monocytes chemoattractant protein-1 functions as a chemoattractant by binding to its receptor in monocytes and macrophages [20]. Several reports demonstrated the role of MCP-1 in various chronic inflammatory conditions, such as atherosclerosis, rheumatoid arthritis, glomerulonephritis, pulmonary hypertension, and pulmonary fibrosis, in human patients [21]. In dogs, MCP-1 has been evaluated in different biological substrates, such as blood [22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37], urine [38, 39], cerebrospinal fluid [40,41,42], synovial fluid [43,44,45], and bronchoalveolar lavage [28]. Most veterinary studies have shown the utility of measuring serum MCP-1 as an inflammatory marker in many inflammatory diseases, such as primary immune-mediated hemolytic anemia, pemphigus foliaceus, and suspected pancreatitis [23, 32, 35], neoplastic diseases, such as lymphoma, hemangiosarcoma, and urothelial carcinoma [24, 36, 39], and infectious diseases, such as babesiosis due to Babesia canis and Babesia rossi, and coccidiomycosis [30, 31, 34]. In human medicine, urinary MCP-1 (uMCP-1) has been associated with kidney damage and inflammation in acute and chronic renal diseases [46,47,48,49,50]. Previous data showed a correlation between elevated uMCP-1 and inflammation represented by macrophages in the renal tissue in human patients [51]. However, there is scarce information on the use of urinary MCP-1 in the detection of acute or chronic kidney damage in dogs [38, 39, 52].

Various human studies have evaluated the usefulness of MCP-1 in cutaneous and visceral leishmaniosis [14, 53,54,55,56,57]. A human study evaluated serum MCP-1 in patients with cutaneous leishmaniosis [58] and only one study evaluated uMCP-1 in patients with visceral leishmaniosis and showed an increase in uMCP-1 normalized to creatinine in those patients compared with healthy patients [59]. In CanL, some studies have evaluated MCP-1 as a marker of the immune response against Leishmania. Strauss-Ayali et al. evaluated the expression of MCP-1 and other chemokines in the spleen in naturally and experimentally L. infantum infected dogs [60]. Some authors showed higher expression of MCP-1 and other chemokines on the skin of dogs with visceral leishmaniosis [61]. Another study evaluated the expression of MCP-1 in the liver and spleen of dogs with visceral leishmaniosis [62]. In 2021, Verçosa and colleagues evaluated apoptosis and MCP-1 expression in renal tissues of Leishmania-infected dogs [63]. Interestingly, an elevation of MCP-1 messenger RNA in renal tissues has recently been studied in CanL, which is associated with infection in dogs from Brazil [64].

Unfortunately, to the best knowledge of the authors, the determination of serum MCP-1 and uMCP-1 in dogs with leishmaniosis has not yet been documented. For these reasons, the aims of the present study were: (1) to determine the serum MCP-1 and the uMCP-1-to-creatinine ratio (uMCP-1/Cr) in healthy dogs and in dogs with leishmaniosis in different clinical stages of the disease [according to the LeishVet and International Renal Interest Society (IRIS) stagings] [65,66,67] using a commercial enzyme-linked immunosorbent assay (ELISA); (2) to assess whether serum MCP-1 and uMCP-1/Cr can differentiate the severity of the disease (based on the LeishVet classification) at the time of diagnosis; and (3) to evaluate the correlation between serum MCP-1 and various inflammatory and renal biomarkers, and the correlation between uMCP-1/Cr and various renal and urinary biomarkers at diagnosis.

Methods

Dogs

This is a cross-sectional study that includes 57 client-owned dogs that were admitted to the San Marco Veterinary Clinic (Veggiano, Italy) for various medical reasons between May and October 2023.

Two study groups were defined as healthy dogs (n = 19) and dogs with leishmaniosis (n = 38). The following inclusion criteria were required to be considered healthy dogs: (1) unremarkable physical examination; (2) normal results in all laboratory tests, including complete blood count (CBC), serum biochemistry, coagulation profile, and urinalysis; (3) a negative L. infantum serology; (4) no history of recent illness; and (5) no drug administration at the time of evaluation. The diagnosis of clinical leishmaniosis was based on compatible clinical signs, clinicopathological findings, a positive L. infantum ELISA serology, and a positive Leishmania real-time polymerase chain reaction (q-PCR) in the bone marrow [2, 65]. All dogs were diagnosed for the first time with clinical leishmaniosis. The following inclusion criteria were required for dogs with leishmaniosis: (1) never treated with conventional anti-Leishmania drugs or immunomodulators, such as domperidone or nucleotides and AHCC or vaccine against leishmaniosis; (2) routine laboratory tests including CBC, serum biochemistry, coagulation profile, urinalysis, and abdominal ultrasound; (3) absence of Dirofilaria immitis antigen (Filarcheck 96, biopronix by Agrolabo, Italy), absence of Anaplasma phagocytophilum, Ehrlichia canis, and Rickettsia conorii antibodies (semiquantitative immunofluorescence by MegaFLUO ANAPLASMA ph. MEGACOR; MegaFLUO EHRLICHIA canis MEGACOR; MegaFLUO RICKETTSIA conorii MEGACOR; Hörbranz, Austria); (4) inactive urine sediment; (5) no other concurrent diseases; and (6) no medication administration in the previous 3 months with the exception of repellent or antiparasitic drugs.

During physical examination, after an adequate acclimatization period to the environment, in all dogs enrolled in the study, systolic blood pressure (SBP) was measured with the automated blood pressure monitor for companion animals SunTech Vet 20 (SunTech Medical Inc., USA), and the average value of four consecutive measurements was recorded. Laboratory blood and urine tests were carried out in the morning after 12 h of fasting without pharmacological or other restrictions.

Once the diagnosis of leishmaniosis was made, all dogs were classified according to LeishVet classification [2, 65, 66], and the recommendations of the International Renal Interest Society (IRIS) for chronic kidney disease [67]. Subsequently, dogs were divided into group 1 if they belong to LeishVet stage IIa or IIb (moderate disease), and group 2 if they belong to LeishVet stage III or IV (severe to very severe disease). According to IRIS staging, after classifying each dog in the stage to which it belongs, dogs were considered in IRIS stage I or aggregated in IRIS stages II + III + IV for statistical analysis.

Blood tests

All tests were performed at the San Marco Veterinary Laboratory (Veggiano, Italy). A blood sample was collected by cephalic or saphenous or jugular venipuncture in a 10 ml sterile plastic syringe. Then, 2 ml of blood was transferred to plastic tubes containing K3-EDTA for a CBC performed on an automated hematology analyzer (ADVIA 2120i, Siemens, Germany) with a blood smear reading. A total of 4 ml of blood was placed in serum glass tubes for chemistry analysis performed in an automated biochemical analyzer (Atellica® Solution, Siemens, Germany), and 2 ml of blood was placed in plastic tubes containing 3.2% sodium citrate for coagulation profile performed in an automated coagulation analyzer (BCSXP, Siemens, Germany). The following parameters were evaluated: paraoxonase-1 (PON-1), haptoglobin (Hp), ferritin (Ft), C-reactive protein (CRP), total iron binding capacity (TIBC), iron, albumin (Alb), globulins (Glob), urea, and creatinine (Cr). In addition, symmetric-dimethylarginine (SDMA) was measured with a canine SDMA ELISA (Eurolyser Diagnostica GmbH, Salzburg, Austria).

To detect L. infantum antibodies, a Leishmania ELISA test was performed following the manufacturer’s instructions (VetLine Leishmania, Leishmania ELISA test, NovaTec Immunodiagnostica GmbH, Dietzenbach, Germany) [68]. The result of the Leishmania ELISA test was considered negative if the antibody level was < 9%, doubtful if the antibody level was 9–11%, and positive if the antibody level was > 11%.

Urine examination

Urine was collected at the time of the visit (in the morning after blood sampling) by free catch in a sterile container in all dogs. A total volume of 10 ml of urine was obtained during spontaneous urination, and 7 ml of urine was used for urinalysis and urinary chemistry performed on an automated urine analyzer (CLINITEK Novus®, Siemens, Germany) and on an automated biochemical analyzer (Atellica® Solution, Siemens, Germany), respectively. Whole urine was used for urine analysis with test strips (CLINITEK Novus Pro12 Urinalysis Cassette, Siemens, Germany), urine specific gravity measurement (USG) (CLINITEK Novus Pro12 Urinalysis Cassette, Siemens, Germany), urine protein to creatinine ratio (UPC) determination (calculated by dividing the concentration of urinary proteins by the concentration of urinary Cr concentration), and urinary chemistry. Urine proteins (UPs) were measured in an automated spectrophotometer (Atellica® Solution, Siemens, Germany) using pyrogallol red (Atellica CH Urinary/Cerebrospinal Fluid Protein (UCFP), Siemens Healthcare Diagnostics Inc., USA), and uCr with a modified Jaffe method (Siemens Healthcare Diagnostics Inc., USA). Samples were automatically prediluted 1:5 to fit the linearity of the method according to manufacturer’s instructions. Urinary sediment was examined by a clinical pathologist with an optical microscope and only dogs with an inactive urine sediment [< 5 white blood cells per high power field (hpf), < 5 red blood cells/hpf or no visible bacteria] were considered for urinary podocin and nephrin measurements. The following parameters in the urine were evaluated: USG, UPC, urinary amylase to creatinine ratio (uAm/Cr), and urinary creatinine (uCr) concentration.

MCP-1 determination

After centrifugation of blood and urine, serum and urinary supernatant were aliquoted in plastic cryotubes, frozen within 2 h after collection and stored at −80 °C until a canine CCL2/MCP-1 Quantikine ELISA Kit (R&D System, Minneapolis, MN, USA) was performed [69]. Once all samples were collected, the ELISA tests to detect serum and urinary MCP-1 were performed according to the manufacturer’s instructions. Briefly, an ELISA plate was set for control, standards, and sample wells. At first, 50 μL of the RD1W assay diluent was added to each well; subsequently, 50 μL of standard, control, or sample per well was added and left to incubate for 2 h at room temperature. After incubation, each well was aspirated and washed with 400 μL of wash buffer for a total of five washes. Then, 100 μL of canine MCP-1 conjugate was added to each well, incubated for 2 h at room temperature, and, again, after incubation, each well was aspirated and washed a total of five times. At this point, 100 μL of substrate solution was added to each well and incubated (protected from light) for 30 min at room temperature. At the end of the incubation, 100 µL of stop solution was added to each well to terminate the reaction. The optical density was determined with a microplate reader at a wavelength of 450 nm in 30 min. The standard curve for MCP-1 started from 1000 pg/mL and two-fold dilutions were made until 15.6 to pg/ml was obtained. Each sample was measured in duplicate, and the average values obtained were expressed in pg/ml. The standard curve was calculated using a computer-generated spline logistic curve fit with programme test result by the automatic analyzer Stratego (Futurlab ®, Limena, Padova, Italy). Once the concentration of uMCP-1 was determined, its levels were assessed relative to the concentration of uCr as uMCP-1/Cr to correct for dilution as reported in previous studies [70, 71].

Evaluation of Leishmania parasitic load

Leishmania q-PCR was measured in the bone marrow of all dogs with leishmaniosis. Bone marrow aspirates were obtained from costochondral junctions using an 18-gauge needle connected to a 10 ml syringe according to the protocol described by Paparcone et al. for the diagnosis of CanL [72] DNA extraction was performed using a High Pure PCR Template Preparation Kit (Roche Science Applied) and carried out according to the manufacturer’s protocol. Real-time PCR was performed using LightCycler FastStart DNA MasterPLUS hybridization probes (Roche, Mannheim, Germany), using a LightCycler version 3.5.17 instrument (Roche, Mannheim, Germany). Commercial L. infantum primers and LC set hybridization probes (TIB Molbiol, Genova, Italy) that amplified a fragment of the kinetoplast minicircle were used. The thermal cycling was performed according to the manufacturer’s instructions (TIB Molbiol). Positive and negative controls were used in all q-PCR runs as previously reported [73].To be considered positive > 100 copies of kinetoplast/ml should be detected in bone marrow.

Statistical analysis

Qualitative data were summarized using percentages. Quantitative variables were reported as mean and standard deviation (SD) or as median and interquartile range (IQR), according to the Shapiro–Wilk’s test for normality. Statistical differences between two groups were analyzed with Student’s t-test, under the hypotheses of normality and homoscedasticity, or with the Mann–Whitney rank test for nonnormal variables. For more than two groups, parametric or nonparametric analysis of variance (ANOVA) was applied, and post hoc analyses were performed through pairwise comparisons between group levels with Holm correction for multiple tests. The relationships between quantitative variables were evaluated using correlation plots. Finally, the receiver-operating characteristic (ROC) curve and the area under the ROC curve (AUC) were used to assess the ability of quantitative parameters to differentiate between the dogs in group 1 and the dogs in group 2 at the time of diagnosis. The accuracy of the test was classified as excellent (AUC > 0.9), good (0.8 < AUC ≤ 0.9), fair (0.7 < AUC ≤ 0.8), poor (0.6 < AUC ≤ 0.7), or failed (0.5 < AUC ≤ 0.6) [74]. The Youden procedure was applied to determine the best threshold value. The significance level was set at P < 0.05. Data were analyzed using the statistical software R version 4.3.2.

Results

The results of demographic and clinical data, and the results of blood and urine analysis of all dogs are reported in Tables 1, 2, and 3. All data supporting the main conclusions are displayed in Additional File 1 (Dataset S1: signalment, clinical data, and serum and urinary parameters including MCP-1).

Table 1 Demographic and clinical data of healthy dogs and dogs with leishmaniosis
Full size table
Table 2 Serum biochemistry results of healthy dogs and dogs with leishmaniosis
Full size table
Table 3 Urine analysis results of healthy dogs and dogs with leishmaniosis
Full size table

A significant increase in respiration rate was found in dogs with leishmaniosis compared with healthy dogs even if the majority of leishmaniotic dogs had a respiration rate in the reference interval (Mann–Witney U-test, U = 237.5, P = 0.04, Table 1).

A significant increase in CRP, Ft, Hp, globulin, and SDMA and a decrease in albumin, iron, and TIBC was observed in leishmaniosis dogs compared with healthy dogs (U = 57.5, P < 0.0001; U = 65.5, P < 0.0001; U = 92, P < 0.0001; U = 72, P < 0.0001; U = 133.5, P < 0.0001; U = 634.5, P < 0.0001; U = 569.5, P < 0.0001; U = 561.5, P < 0.0001, respectively, Table 2). Serum MCP-1 was significantly higher in dogs with leishmaniosis compared with healthy dogs (U = 62, P < 0.0001, Table 2).

The UPC, uAm/Cr, uMCP-1, and the uMCP-1/Cr increased significantly in dogs with leishmaniosis compared with healthy dogs (U = 57.5, P = 0.0003; U = 57, P < 0.0001; U = 149, P = 0.0002; U = 90, P < 0.0001, respectively, Table 3).

According to the LeishVet guidelines, dogs with leishmaniosis were classified as stage IIa (n = 12), stage IIb (n = 7), stage III (n = 10), and stage IV (n = 9). Subsequently, all dogs were divided into group 1 (stages IIa and IIb, n = 19) and group 2 (stage III and IV, n = 19).

According to the IRIS staging of chronic kidney disease, dogs were classified as stage I (n = 31), stage II (n = 4), stage III (n = 1), and stage IV (n = 2), and subsequently aggregated as stage I (n = 31) and as stages II + III + IV (n = 7).

Higher serum MCP-1 was observed in dogs in Leishvet stage IIb [median values and interquartile range (IQR): 455.9 pg/ml (290.17), Fig. 1A], III (median values and IQR: 357.6 pg/ml (246.76), Fig. 1A), and IV (median values and IQR: 784.9 pg/ml (664.24); Fig. 1A) compared with healthy dogs (median values and IQR: 124.83 pg/ml (48.05); Kruskall–Wallis H-test, H = 38.57, df = 4, P < 0.0001, respectively, Fig. 1A). Serum MCP-1 was also significantly higher in dogs in stage IV of Leishvet than in dogs in stage IIa (median values and IQR: 784.9 pg/ml (664.24) versus 280.65 pg/ml (197.7); post hoc U = 107, P < 0.0001, Fig. 1A). Increasing values were found in dogs in Leishvet stage IIa compared with healthy dogs and in dogs in stage IIb compared with dogs in stage IIa, but the difference was not statistically significant (post hoc U = 70, P = 0.16; post hoc U = 107, df = 4, P = 0.1, respectively, Fig. 1A).

Fig. 1
figure 1

Box plots showing A serum MCP-1 and B uMCP-1/Cr in healthy dogs and dogs with leishmaniosis according to the LeishVet clinical staging system to which they belong. Stage IIa, LeishVet stage IIa; Stage IIb, LeishVet stage IIb; Stage III, LeishVet stage III; and Stage IV, LeishVet stage IV. *Statistically significant difference between the median of each group

Full size image

Higher uMCP-1/Cr was observed in dogs in LeishVet stage IIb (median values and IQR: 9.92 × 10−7 (10.86 × 10−7), Fig. 1B), III (median values and IQR: 13.03 × 10−7 (9.33 × 10−7), Fig. 1B), and IV (median values and IQR: 24.19 × 10−7 (17.99 × 10−7), Fig. 1B) compared with healthy dogs (median values and interquartile range (IQR): 1.21 × 10−7 (2.08 × 10−7); H = 34.91, df = 4, P = 0.027, P < 0.0001, P < 0.0001, respectively, Fig. 1B). Furthermore, dogs in stages III and IV of LeishVet (median values and IQR: 13.03 × 10−7 (9.33 × 10−7) and 24.19 × 10−7 (17.99 × 10−7), respectively, Fig. 1B) had significantly higher uMCP-1/Cr compared with dogs in stage IIa (median values and IQR: 2.86 × 10−7 (2.04 × 10−7); post hoc U = 110, P = 0.003; post hoc U = 108, P < 0.0001, respectively, Fig. 1B). Increasing values were found in dogs in LeishVet stage IIa compared with healthy dogs and in dogs in stage IIb, but the difference was not statistically significant (post hoc U = 62, P = 0.18; post hoc U = 63, P = 0.28, respectively, Fig. 1B).

According to IRIS stage, a higher serum MCP-1 was observed in dogs in stage I (median values and IQR: 331 pg/ml (247.27), Fig. 2A), and in dogs in stages II + III + IV (median values and IQR: 775.3 pg/ml (748.01), Fig. 2A) compared with healthy dogs (median values and IQR: 124.83 pg/ml (48.05); H = 28.55, df = 2, P < 0.0001, Fig. 2A). Furthermore, serum MCP-1/Cr was significantly higher in dogs in stages II + III + IV compared with dogs in stage I (H = 28.55, df = 2, P < 0.0001, Fig. 2A).

Fig. 2
figure 2

Box plots showing A serum MCP-1 and B uMCP-1/Cr in healthy dogs and dogs with leishmaniosis according to the IRIS staging to which they belong. *Statistically significant difference between the median of each group.

Full size image

Dogs with higher uMCP-1/Cr were detected in stage I and in stages II + III + IV of IRIS (median values and IQR: 4.27 × 10−7 (17.99 × 10−7) and 10.75 × 10−7 (16.31 × 10−7), respectively, Fig. 2B) compared to healthy dogs (median and IQR: 1.21 × 10−7 (11.59 × 10−7); H = 22.51, df = 2, P < 0.0001, respectively, Fig. 2B). Despite the higher values of uMCP-1/Cr in dogs’ stages II + III + IV of IRIS compared with dogs in stage I, the difference was not statistically significant (H = 22.51, df = 2, P = 0.18, Fig. 2B).

ROC curves were calculated to differentiate between dogs with leishmaniosis with moderate disease (group 1) from those with severe to very severe disease (group 2) at the time of diagnosis. Serum MCP-1 showed a fair accuracy (AUC = 0.78, 95% CI 0.64–0.93; P = 0.002, Fig. 3A) and the threshold value was 336.85 with a sensitivity of 79% and a specificity of 68%. uMCP-1/Cr showed a good accuracy (AUC = 0.86, 95% CI 0.74–0.99; P < 0.0001, Fig. 3B), and the threshold value was 6.9 × 10−7 with a sensitivity of 84% and a specificity of 79%. Of all variables studied, uAm/Cr was the best parameter to detect the severity of the disease with statistical significance at the time of diagnosis (Table 8). On the basis of the ROC curves, uMCP-1/Cr, TIBC, and Alb were shown to be good markers to discriminate between group 1 and group 2 (Table 4; Fig. 3B).

Fig. 3
figure 3

Receiver operating characteristic curve and area under the curve (AUC) to differentiate between dogs with leishmaniosis with moderate disease (LeishVet IIa + IIb) and with severe to very severe disease (LeishVet III + IV) using A serum MCP-1 and B uMCP-1/Cr. Younden index cut-off with their associated sensitivity (Se) and specificity (Sp) is presented for each curve

Full size image
Table 4 Area under the curve of various variables between group 1 and group 2
Full size table

In healthy dogs, there was a weak positive correlation between serum MCP-1 and uMCP-1/Cr (Pearson’s coefficient correlation, r = 0.22, P < 0.001), while in sick dogs with leishmaniosis, a moderate positive correlation between these two markers was found (r = 0.42, P < 0.0001).

There was no significant correlation between serum MCP-1 and Alb in healthy dogs (r = 0.19, P = 0.43) and a moderate negative correlation in sick dogs (r = −0.43; P = 0.007). When considering the association between serum MCP-1 and Glob, a positive correlation was found in healthy dogs (r = 0.52, P = 0.02) and no correlation was found in dogs with leishmaniosis (r = 0.16, P = 0.35, respectively).

In healthy dogs, no significant correlation was found between serum MCP-1 and various and renal markers except urea (r = 0.54, P < 0.0005), while a significant positive correlation between serum MCP-1 and inflammatory markers, such as iron and TIBC, was found (r = 0.47, P < 0.005; r = 0.61, P < 0.0005, respectively, Table 5). In dogs with leishmaniosis there was a moderate positive correlation between serum MCP-1 and CRP, urea, Cr, and SDMA (r = 0.49, P < 0.0005; r = 0.50, P < 0.0001; r = 0.47, P < 0.005; r = 0.49, P < 0.0005, respectively) and a negative correlation with TIBC (r = −0.42, P < 0.0005, Table 6).

Table 5 Correlation between serum MCP-1 and various inflammatory and renal markers in healthy dogs
Full size table
Table 6 Correlation between serum MCP-1 and various inflammatory and renal markers in leishmaniotic dogs
Full size table

In healthy dogs, there was a moderate negative correlation between the uMCP-1/Cr and USG (r = −0.45, P < 0.0005, Table 7). When considering dogs with leishmaniosis, a negative correlation was still present between the uMCP-1/Cr and USG (r = −0.49, P < 0.0005) and a strong positive correlation between the uMCP-1/Cr and UPC (r = 0.72, P < 0.0005), and uMCP-1/Cr and uAm/Cr was observed (r = 0.74, P < 0.0005, Table 8). Interestingly, there was also a positive correlation between the uMCP-1/Cr with SDMA but not with Cr (r = 0.44, P < 0.0005; r = 0.17, P = 0.30, respectively, Table 8). Furthermore, a strong positive correlation and a very strong positive correlation were detected between Cr and SDMA and UPC and uAm/Cr (r = 0.72, P < 0.0005, r = 0.91, P < 0.0005, respectively, Table 8).

Table 7 Correlation between the uMCP-1 and various urinary and renal markers in healthy dogs
Full size table
Table 8 Correlation between the uMCP-1 and various urinary and renal markers in leishmaniotic dogs
Full size table

Discussion

This study described, for the first time, serum MCP-1 and uMCP-1 measurements in dogs with leishmaniosis by using an ELISA and showed a significantly increased serum MCP-1 and uMCP-1/Cr in dogs with leishmaniosis compared with healthy dogs. Monocyte chemoattractant protein-1 plays an important role in the pathophysiology of L. infantum infection and various veterinary studies have investigated the expression of MCP-1 in visceral organs and skin of dogs with clinical leishmaniosis [60,61,62,63,64]. The result of these previous studies was an increase in MCP-1 expression in different tissues associated with disease progression in agreement with the present study, suggesting an accumulation of infiltrating macrophages attracted by MCP-1 as a response of the immune system [60, 61]. Serum MCP-1 in dogs with leishmaniosis was increased, unlike that found in a recent human study in which serum MCP-1, evaluated with ELISA, in patients with cutaneous leishmaniosis was lower compared with the healthy control group [58]. The different result between the two studies probably had multiple reasons: first, the different clinical form of the disease, dogs with leishmaniosis had mainly systemic manifestation of L. infantum infection in contrast with human patients, which showed only localized cutaneous lesions owing to L. major or L. tropica infection. Therefore, in humans, serum MCP-1 is not elevated likely due to the fact the inflammatory process is localized and not systemic while in dogs the inflammatory process is systemic and it is well known that the immune response to infection partially depends on the specific Leishmania spp. involved and its virulence; and second, a considerable different host dependent component among dogs and human patients.

The increase in uMCP-1/Cr in dogs with leishmaniosis compared with healthy dogs was consistent with the results of a human study in which, at diagnosis, patients with visceral leishmaniosis had a higher uMCP-1/Cr compared with the healthy control, suggesting the presence of inflammation and incipient renal damage in the kidneys [59]. The common result can be explained by the fact that in both studies there was parasite dissemination after infection. When considering dogs with leishmaniosis based on LeishVet clinical staging and IRIS staging, dogs in LeishVet stage IIb and IRIS stage I had significantly higher serum MCP-1 and uMCP-1/Cr compared with healthy dogs. These data are important because they show the presence of systemic inflammation on one side and renal inflammation and damage on the other before a change in serum Cr, which is considered one of the main biomarkers of a reduction in renal function. Oliveira and colleagues also showed an increase in uMCP-1/Cr in the absence of an increase in serum Cr in humans, suggesting that inflammation and incipient renal damage can occur before changes in Cr in human patients with visceral leishmaniosis [59].

Interestingly, according to the clinical staging of LeishVet and the staging of IRIS, serum MCP-1 was significantly higher in dogs in stage IV of LeishVet compared with stage IIa and in dogs in stage II + III + IV of IRIS compared with stage I. These results are in line with those of a study in dogs with B. canis infection in which complicated dogs (and therefore dogs with more severe disease) had higher serum MCP-1 compared with uncomplicated dogs [30]. The dogs in LeishVet stage III (dogs with severe disease) had similar serum MCP-1 compared with dogs in LeishVet stage IIa and IIb (dogs with moderate disease), even if there was a decreasing trend in dogs in Leishvet stage IIa compared with dogs in LeishVet stage III and, an increasing trend in dogs in LeishVet stage IIb compared with dogs in LeishVet stage III. This result was surprising because a higher level of serum MCP-1 would be expected in dogs with more severe disease, but the difference in the sum of ranks was not large enough to be statistically significant at the alpha equals 0.05 level.

uMCP-1/Cr was significantly higher in dogs in stages III and IV of LeishVet compared with stage IIa. These results can be explained by the fact that MCP-1 is produced by intrinsic renal cells, mesangial, and endothelial cells, when stimulated by inflammatory inducers, such as immune complexes [75], which are more commonly encountered in dogs with leishmaniosis with more advanced stages of renal disease [65]. A recent study, evaluating collagen deposition in the kidneys caused by chronic renal inflammation secondary to leishmaniosis, showed that in dogs with leishmaniosis collagen deposition is linked to different cytokines / chemokines differentially expressed in renal tissue; among the various chemokines studied, the authors showed that MCP-1 expression levels were higher in clinically affected dogs compared with subclinical infected dogs [64]. These data seem to support the results of the present study, although it should be considered that Verçosa et al. evaluated the expression of MCP-1 in the kidneys but did not measure MCP-1 in urine [64].

Serum MCP-1 and uMCP-1/Cr were also evaluated as possible markers to discriminate the severity of the disease according to the clinical staging of LeishVet. On the basis of the analysis of the ROC curve, uMCP-1/Cr and serum MCP-1 showed a good and a fair accuracy in differentiating dogs with moderate disease (stages IIa and IIb) from dogs with severe to very severe disease (stages III and IV). These results confirmed the critical role of MCP-1 in the pathogenesis of kidney disease and the progression of kidney disease and that, especially uMCP-1/Cr, can help diagnose the severity of the disease. Among the various biomarkers studied, uAm/Cr showed excellent accuracy in discriminating between dogs with moderate disease and dogs with severe to very severe disease and, therefore it was the best marker to show the severity of leishmaniosis. Urinary MCP-1/Cr, TIBC, and Alb were also useful markers to differentiate dogs with moderate disease from dogs with severe to very severe disease. These results underlined how urinary markers, first and foremost inflammatory markers, played a fundamental role in showing the severity of leishmaniosis in dogs before changes in classic biomarkers of renal function, such as Cr. Another important aspect to consider is that although leishmaniosis is primarily a glomerular disease in dogs [76, 77], tubulointerstitial damage also occurs during the course of the disease, and uMCP-1/Cr could be used as an early biomarker of tubulointerstitial and glomerular damage as previously described [78].

In the present study, a correlation analysis was performed to evaluate whether there was an association between serum MCP-1 and various inflammatory and renal parameters. A positive correlation was found between serum MCP-1 and CRP and a negative correlation between serum MCP-1, Alb, and TIBC, respectively. The positive correlation between serum MCP-1 and CRP has already been described in dogs in various inflammatory diseases, such as inflammatory bowel disease, immune-mediated hemolytic anemia, thrombocytopenia purpura, eosinophilic pneumonia, immune-mediated arthritis, glomerular nephritis, pancreatitis, and panniculitis, in a previous study [27]. Generally, CRP may increase in response to mobilized inflammatory cytokines in acute disorder, while monocytes would mobilize classically in chronic disorder [27]. If the inflammatory stimulus persists over time, the CRP may remain elevated suggesting the presence of an active inflammation [9, 10]. On the basis of the results of the present study, an increase in CRP and serum MCP-1 could be expected to begin from the moderate stages of CanL and, therefore, during the progression of the disease. The negative correlation between serum MCP-1, Alb, and TIBC suggests the potential role of serum MCP-1 as a marker of inflammation during leishmaniosis. In a previous study, a decrease in TIBC with an increase in CRP has been identified as inflammatory markers in dogs with leishmaniosis [79]. Interestingly, in the present study, there was a negative association between TIBC and CRP in sick dogs, but the lack of a strong correlation suggests that these two markers are partially regulated independently, as suggested by Silvestrini et al. [79]. In the current study, there was also a moderate positive correlation between serum MCP-1 and urea, Cr, and SDMA suggesting that serum MCP-1 could be a potential marker of renal disease in dogs with leishmaniosis. To date, the role of serum MCP-1 as renal marker remains to be defined, as shown by the review by Liu et al. in which only few studies evaluated plasma or serum MCP-1 in human kidney diseases as a marker of risk of acute kidney injury or progression of kidney disease [80].

Among the renal function markers evaluated, there was a positive correlation between uMCP-1/Cr and SDMA, a negative correlation between uMCP-1/Cr and USG but, no correlation between uMCP-1/Cr with serum Cr. When considering markers of tubular and/or glomerular damage there was a strong positive correlation between uMCP-1/Cr and UPC and between uMCP-1/Cr and uAm/Cr. These results are very different from those of a human study on visceral leishmaniosis in which they evaluated the correlation between uMCP-1/Cr and Cr, glomerular filtration rate, UPC, and urinary albumin to creatinine ratio and only found a significant association with urinary albumin to creatinine ratio as an early biomarker of glomerular damage [78].

The general results of this study show the clinical utility of serum MCP-1 and uMCP-1/Cr in CanL to detect systemic inflammation and renal damage with the first marker and, renal inflammation and damage with the second, even if uMCP-1/Cr appears to be a better marker to identify renal damage compared with serum MCP-1. There is a need for further investigation to confirm these preliminary observations, to evaluate MCP-1 in serum and urine of healthy seropositive dogs and sick infected dogs with mild disease (LeishVet stage I) as potential markers to identify inflammation and early renal damage in the absence of established renal disease. In the future, it would also be interesting to evaluate serum MCP-1 and uMCP-1/Cr as markers for monitoring response to specific antileishmanial treatment and as prognostic markers in long-term monitoring.

This study has several limitations. The limited number of dogs studied could have reduced statistical power to detect significant differences in the variables studied (especially when the dogs were further divided into different groups according to the LeishVet and IRIS stagings). No kidney biopsy was performed in healthy dogs and dogs with leishmaniosis; therefore, it is not known in which dog there was renal disease and eventually the severity based on specific histopathological changes in the kidneys.

Conclusions

Serum MCP-1 and uMCP-1/Cr were higher in dogs with leishmaniosis compared with healthy dogs. Furthermore, serum MCP-1 and uMCP-1/Cr were more elevated in advanced stages of the disease compared with moderate stages and, therefore can be markers of the severity of the disease process.

Availability of data and materials

All data supporting the main conclusions are available in the manuscript and its associated files.

Abbreviations

Alb:

Albumin

AUC:

Area under the curve

CanL:

Canine leishmaniosis

CBC:

Complete blood count

Cr:

Creatinine

CRP:

C-reactive protein

ELISA:

Enzyme-linked immunosorbent assay

Ft:

Ferritin

Glob:

Globulins

Hp:

Haptoglobin

IQR:

Interquartile range

IRIS:

International Renal Interest Society

L. infantum :

Leishmania infantum

MCP-1:

Monocytes chemoattractant protein-1

PON-1:

Paraoxonase-1

q-PCR:

Real-time polymerase-chain-reaction

ROC:

Receiver-operating characteristic

SBP:

Systolic blood pressure

SDMA:

Symmetric-dimethylarginine

TIBC:

Total iron binding capacity

uAm/Cr:

Urinary amylase to creatinine ratio

uCr:

Urinary creatinine

uMCP-1:

Urinary monocytes chemoattractant protein-1

uMCP-1/Cr:

Urinary monocytes chemoattractant protein-1 to creatinine ratio

UPC:

Urine protein to creatinine ratio

USG:

Urine specific gravity

References

  1. Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol. 2009;165:1–18.

    Article  CAS  PubMed  Google Scholar 

  2. Solano-Gallego L, Miró G, Koutinas A, Cardoso L, Pennisi MG, Ferrer L, et al. LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vectors. 2011;4:1–16.

    Article  Google Scholar 

  3. Koutinas AF, Polizopoulou ZS, Saridomichelakis MN, Argyriadis D, Fytianou A, Plevraki KG. Clinical considerations on canine visceral leishmaniasis in Greece: a retrospective study of 158 cases (1989–1996). J Am Anim Hosp Assoc. 1999;35:376–83.

    Article  CAS  PubMed  Google Scholar 

  4. Martínez-Subiela S, Tecles F, Eckersall PD, Cerón JJ. Serum concentrations of acute phase proteins in dogs with leishmaniasis. Vet Rec. 2002;150:241–4.

    Article  PubMed  Google Scholar 

  5. Martinez-Subiela S, Strauss-Ayali D, Cerón JJ, Baneth G. Acute phase protein response in experimental canine leishmaniosis. Vet Parasitol. 2011;180:197–202.

    Article  CAS  PubMed  Google Scholar 

  6. Martinez-Subiela S, Cerón JJ, Strauss-Ayali D, Garcia-Martinez JD, Tecles F, Tvarijonaviciute A, et al. Serum ferritin and paraoxonase-1 in canine leishmaniosis. Comp Immunol Microbiol Infect Dis. 2014;37:23–9.

    Article  CAS  PubMed  Google Scholar 

  7. Escribano D, Tvarijonaviciute A, Kocaturk M, Cerón JJ, Pardo-Marín L, Torrecillas A, et al. Serum apolipoprotein-A1 as a possible biomarker for monitoring treatment of canine leishmaniosis. Comp Immunol Microbiol Infect Dis. 2016;49:82–7.

    Article  PubMed  Google Scholar 

  8. Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med. 1999;340:448–54.

    Article  CAS  PubMed  Google Scholar 

  9. Cerón JJ, Martinez-Subiela S, Ohno K, Caldin M. A seven-point plan for acute phase protein interpretation in companion animals. Vet J. 2008;177:6–7.

    Article  PubMed  Google Scholar 

  10. Eckersall PD, dos Schmidt EMS. The final hurdles for acute phase protein analysis in small animal practice. J Small Anim Pract. 2014;55:1–3.

    Article  PubMed  Google Scholar 

  11. McMahon-Pratt D, Alexander J. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunol Rev. 2004;201:206–24.

    Article  PubMed  Google Scholar 

  12. Müller K, Zandbergen G, Hansen B, Laufs H, Jahnke N, Solbach W, et al. Chemokines, natural killer cells and granulocytes in the early course of Leishmania major infection in mice. Med Microbiol Immunol. 2001;190:73–6.

    Article  PubMed  Google Scholar 

  13. Rot A, Von Andrian UH. Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells. Annu Rev Immunol. 2004;22:891–928.

    Article  CAS  PubMed  Google Scholar 

  14. Ritter U, Körner H. Divergent expression of inflammatory dermal chemokines in cutaneous leishmaniasis. Parasite Immunol. 2002;24:295–301.

    Article  CAS  PubMed  Google Scholar 

  15. Toepp AJ, Petersen CA. The balancing act: immunology of leishmaniosis. Res Vet Sci. 2020;130:19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Barna BP, Pettay J, Barnett GH, Zhou P, Iwasaki K, Estes ML. Regulation of monocyte chemoattractant protein-1 expression in adult human non-neoplastic astrocytes is sensitive to tumor necrosis factor (TNF) or antibody to the 55-kDa TNF receptor. J Neuroimmunol. 1994;50:101–7.

    Article  CAS  PubMed  Google Scholar 

  17. Birdsall HH, Green DM, Trial JA, Youker KA, Burns AR, MacKay CR, et al. Complement C5a, TGF-beta 1, and MCP-1, in sequence, induce migration of monocytes into ischemic canine myocardium within the first one to five hours after reperfusion. Circulation. 1997;95:684–92.

    Article  CAS  PubMed  Google Scholar 

  18. Brown Z, Strieter RM, Neild GH, Thompson RC, Kunkel SL, Westwick J. IL-1 receptor antagonist inhibits monocyte chemotactic peptide 1 generation by human mesangial cells. Kidney Int. 1992;42:95–101.

    Article  CAS  PubMed  Google Scholar 

  19. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, et al. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. PNAS. 1990;87:5134–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Balkwill F. Chemokine biology in cancer. Semin Immunol. 2003;15:49–55.

    Article  CAS  PubMed  Google Scholar 

  21. Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009;29:313–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Duffy AL, Olea-Popelka FJ, Eucher J, Rice DM, Dow SW. Serum concentrations of monocyte chemoattractant protein-1 in healthy and critically ill dogs. Vet Clin Pathol. 2010;39:302–5.

    Article  PubMed  Google Scholar 

  23. Kjelgaard-Hansen M, Goggs R, Wiinberg B, Chan DL. Use of serum concentrations of interleukin-18 and monocyte chemoattractant protein-1 as prognostic indicators in primary immune-mediated hemolytic anemia in dogs. J Vet Intern Med. 2011;25:76–82.

    Article  CAS  PubMed  Google Scholar 

  24. Perry JA, Thamm DH, Eickhoff J, Avery AC, Dow SW. Increased monocyte chemotactic protein-1 concentration and monocyte count independently associate with a poor prognosis in dogs with lymphoma. Vet Comp Oncol. 2011;9:55–64.

    Article  CAS  PubMed  Google Scholar 

  25. O’Neill S, Drobatz K, Satyaraj E, Hess R. Evaluation of cytokines and hormones in dogs before and after treatment of diabetic ketoacidosis and in uncomplicated diabetes mellitus. Vet Immunol Immunopathol. 2012;148:276–83.

    Article  PubMed  Google Scholar 

  26. Zois NE, Moesgaard SG, Kjelgaard-Hansen M, Rasmussen CE, Falk T, Fossing C, et al. Circulating cytokine concentrations in dogs with different degrees of myxomatous mitral valve disease. Vet J. 2012;192:106–11.

    Article  CAS  PubMed  Google Scholar 

  27. Ishioka K, Suzuki Y, Tajima K, Ohtaki S, Miyabe M, Takasaki M, et al. Monocyte chemoattractant protein-1 in dogs affected with neoplasia or inflammation. J Vet Med Sci. 2013;75:173–7.

    Article  CAS  PubMed  Google Scholar 

  28. Roels E, Krafft E, Farnir F, Holopainen S, Laurila HP, Rajamäki MM, et al. Assessment of CCL2 and CXCL8 chemokines in serum, bronchoalveolar lavage fluid and lung tissue samples from dogs affected with canine idiopathic pulmonary fibrosis. Vet J. 2015;206:75–82.

    Article  CAS  PubMed  Google Scholar 

  29. Goggs R, Letendre JA. High mobility group Box-1 and pro-inflammatory cytokines are increased in dogs after trauma but do not predict survival. Front Vet Sci. 2018;5:179.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Galán A, Mayer I, Rafaj RB, Bendelja K, Sušić V, Cerón JJ, et al. MCP-1, KC-like and IL-8 as critical mediators of pathogenesis caused by Babesia canis. PLoS One. 2018;e0190474.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Celliers A, Rautenbach Y, Hooijberg E, Christopher M, Goddard A. Neutrophil myeloperoxidase index in dogs with babesiosis caused by Babesia rossi. Front Vet Sci. 2020;7:72.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Choi SW, Kim YH, Kang MS, Jeong Y, Ahn JO, Choi JH, et al. Serum concentration of inflammatory cytokines in dogs with suspected acute pancreatitis. Vet Sci. 2021;8:51.

    PubMed  PubMed Central  Google Scholar 

  33. Szczubia M, Łopuszyski W, Dabrowski R, Jamio M, Bochniarz M, Brodzki P. Plasma levels of chemokines CCL2 and CXCL12 in female dogs with malignant mammary gland tumours without and with metastases. Pol J Vet Sci. 2023;26:385–92.

    Article  Google Scholar 

  34. Jaffey JA, Shubitz LF, Johnson MDL, Bolch CA, da Cunha A, Murthy AK, et al. Evaluation of host constitutive and ex vivo coccidioidal antigen-stimulated immune response in dogs with naturally acquired coccidioidomycosis. J Fungi. 2023;9:213.

    Article  CAS  Google Scholar 

  35. Starr H, Howerth E, Gogal R, Barber J, Leon R, Blubaugh A, et al. Characterization of the serum and skin inflammatory profile in canine pemphigus foliaceus using multiplex assay and quantitative real-time polymerase chain reaction (qRT-PCR). Vet Immunol Immunopathol. 2023;262:110631. https://doi.org/10.1016/j.vetimm.2023.110631.

    Article  PubMed  Google Scholar 

  36. Regan DP, Escaffi A, Coy J, Kurihara J, Dow SW. Role of monocyte recruitment in hemangiosarcoma metastasis in dogs. Vet Comp Oncol. 2017;15:1309–22.

    Article  CAS  PubMed  Google Scholar 

  37. Souza CP, Schissler JR, Contreras ET, Dow SW, Hopkins LS, Coy JW, et al. Evaluation of immunological parameters in pit bull terrier-type dogs with juvenile onset generalized demodicosis and age-matched healthy pit bull terrier-type dogs. Vet Dermatol. 2018;29:482-e162.

    Article  PubMed  Google Scholar 

  38. McDuffie JE, Sablad M, Ma JY, Snook S. Urinary parameters predictive of cisplatin-induced acute renal injury in dogs. Cytokine. 2010;52:156–62.

    Article  CAS  PubMed  Google Scholar 

  39. Shimizu N, Hamaide A, Dourcy M, Noël S, Clercx C, Teske E. Evaluation of urinary and serum level of chemokine (C-C motif) ligand 2 as a potential biomarker in canine urothelial tumours. Vet Comp Oncol. 2019;17:11–20.

    Article  CAS  PubMed  Google Scholar 

  40. Martin-Vaquero P, da Costa RC, Moore SA, Gross AC, Eubank TD. Cytokine concentrations in the cerebrospinal fluid of great danes with cervical spondylomyelopathy. J Vet Intern Med. 2014;28:1268–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Taylor AR, Welsh CJ, Young C, Spoor E, Kerwin SC, Griffin JF, et al. Cerebrospinal fluid inflammatory cytokines and chemokines in naturally occurring canine spinal cord injury. J Neurotrauma. 2014;31:1561–9.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Barber RM, Platt SR, De Risio L, Barber J, Robinson KR. Multiplex analysis of cytokines in the cerebrospinal fluid of dogs after ischemic stroke reveals elevations in chemokines CXCL1 and MCP-1. Front Vet Sci. 2023;10:1169617.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Murakami K, Maeda S, Yonezawa T, Matsuki N. CC chemokine ligand 2 and CXC chemokine ligand 8 as neutrophil chemoattractant factors in canine idiopathic polyarthritis. Vet Immunol Immunopathol. 2016;182:52–8.

    Article  CAS  PubMed  Google Scholar 

  44. Kleine SA, Gogal RM, George C, Thaliath M, Budsberg SC. Elevated synovial fluid concentration of monocyte chemoattractant protein-1 and interleukin-8 in dogs with osteoarthritis of the stifle. Vet Comp Orthop Traumatol. 2020;33:147–50.

    Article  PubMed  Google Scholar 

  45. Malek S, Weng HY, Martinson SA, Rochat MC, Béraud R, Riley CB. Evaluation of serum MMP-2 and MMP-3, synovial fluid IL-8, MCP-1, and KC concentrations as biomarkers of stifle osteoarthritis associated with naturally occurring cranial cruciate ligament rupture in dogs. PLoS One. 2020;15:e0242614.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Grandaliano G, Gesualdo L, Ranieri E, Monno R, Montinaro V, Marra F, et al. Monocyte chemotactic peptide-1 expression in acute and chronic human nephritides. J Am Soc Nephrol. 1996;7:906–13.

    Article  CAS  PubMed  Google Scholar 

  47. Hodgkins KS, Schnaper HW. Tubulointerstitial injury and the progression of chronic kidney disease. Pediatr Nephrol. 2012;27:901–9.

    Article  PubMed  Google Scholar 

  48. Mansour SG, Puthumana J, Coca SG, Gentry M, Parikh CR. Biomarkers for the detection of renal fibrosis and prediction of renal outcomes: a systematic review. BMC Nephrol. 2017;18:72. .

    Article  PubMed  PubMed Central  Google Scholar 

  49. Beker BM, Corleto MG, Fieiras C, Musso CG. Novel acute kidney injury biomarkers: their characteristics, utility and concerns. Int Urol Nephrol. 2018;50:705–13.

    Article  CAS  PubMed  Google Scholar 

  50. Thakur V, Chattopadhyay M. Early urinary markers for diabetic and other kidney diseases. Curr Drug Targets. 2018;19:825–31.

    Article  PubMed  Google Scholar 

  51. Eardley KS, Zehnder D, Quinkler M, Lepenies J, Bates RL, Savage CO, et al. The relationship between albuminuria, MCP-1/CCL2, and interstitial macrophages in chronic kidney disease. Kidney Int. 2006;69:1189–97.

    Article  CAS  PubMed  Google Scholar 

  52. Davis J, Raisis AL, Miller DW, Rossi G. Validation of a commercial magnetic bead–based multiplex assay for 5 novel biomarkers of acute kidney injury in canine serum. J Vet Diagn Invest. 2020;32:656–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ritter U, Moll H, Laskay T, Bröcker EB, Velazco O, Becker I, et al. Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis. J Infect Dis. 1996;173:699–709.

    Article  CAS  PubMed  Google Scholar 

  54. Valencia-Pacheco G, Loría-Cervera EN, Sosa-Bibiano EI, Canché-Pool EB, Vargas-Gonzalez A, Melby PC, et al. In situ cytokines (IL-4, IL-10, IL-12, IFN-γ) and chemokines (MCP-1, MIP-1α) gene expression in human Leishmania (Leishmania) mexicana infection. Cytokine. 2014;69:56–61.

    Article  CAS  PubMed  Google Scholar 

  55. Ibarra-Meneses AV, Sanchez C, Alvar J, Moreno J, Carrillo E. Monocyte chemotactic protein 1 in plasma from soluble Leishmania antigen-stimulated whole blood as a potential biomarker of the cellular immune response to Leishmania infantum. Front Immunol. 2017;8:1208.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Singh N, Sundar S. Inflammatory chemokines and their receptors in human visceral leishmaniasis: Gene expression profile in peripheral blood, splenic cellular sources and their impact on trafficking of inflammatory cells. Mol Immunol. 2017;85:111–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kumar R, Bhatia M, Pai K. Role of chemokines in the pathogenesis of visceral leishmaniasis. Curr Med Chem. 2022;29:5441–61.

    Article  CAS  PubMed  Google Scholar 

  58. Hassanshahi G, Alavi SE, Khorramdelazad H, Ahmadi Z, Fattahi Bafghi A, Abdollahi SH, et al. Serum levels of CC chemokine ligands in cutaneous leishmaniasis patients. J Parasit Dis. 2021;45:153.

    Article  PubMed  Google Scholar 

  59. Oliveira MJC, Silva Junior GB, Sampaio AM, Montenegro BL, Alves MP, Henn GAL, et al. Preliminary study on tubuloglomerular dysfunction and evidence of renal inflammation in patients with visceral leishmaniasis. Am J Trop Med Hyg. 2014;91:908.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Strauss-Ayali D, Baneth G, Jaffe CL. Splenic immune responses during canine visceral leishmaniasis. Vet Res. 2007;38:547–64.

    Article  CAS  PubMed  Google Scholar 

  61. Menezes-Souza D, Guerra-Sá R, Carneiro CM, Vitoriano-Souza J, Giunchetti RC, Teixeira-Carvalho A, et al. Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis. PLoS Negl Trop Dis. 2012;6:e1566.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Nascimento MSL, Albuquerque TDR, Do-Valle-Matta MA, Caldas IS, Diniz LF, Talvani A, et al. Naturally Leishmania infantum-infected dogs display an overall impairment of chemokine and chemokine receptor expression during visceral leishmaniasis. Vet Immunol Immunopathol. 2013;153:202–8.

    Article  CAS  PubMed  Google Scholar 

  63. Araújo Verçosa BL, Muniz-Junqueira MI, Menezes-Souza D, Mourão Dias Magalhães L, Fujiwara RT, Melo MN, et al. Enhanced apoptotic index, chemokines and inflammatory recruitment in renal tissues shows relationship with the clinical signs in Leishmania-infected dogs. Vet Parasitol. 2021;300:109611.

    Article  PubMed  Google Scholar 

  64. Verçosa BLA, Muniz-Junqueira MI, Menezes-Souza D, Fujiwara RT, de Borges L, Melo FMN, et al. MCP-1/IL-12 ratio expressions correlated with adventitial collagen depositions in renal vessels and IL-4/IFN-γ expression correlated with interstitial collagen depositions in the kidneys of dogs with canine leishmaniasis. Mol Immunol. 2023;156:61–76.

    Article  PubMed  Google Scholar 

  65. Solano-Gallego L, Cardoso L, Pennisi MG, Petersen C, Bourdeau P, Oliva G, et al. Diagnostic challenges in the Era of canine Leishmania infantum Vaccines. Trends Parasitol. 2017;33:706–17.

    Article  CAS  PubMed  Google Scholar 

  66. LeishVet canine factsheets 2024 for the practical management of canine leishmaniosis - LeishVet. https://www.leishvet-alive.com/wp-content/uploads/2024/04/FS-ALIVE24-canine.pdf.

  67. International Renal Interest Society: IRIS Staging of CKD (modified 2019). International Renal Interest Society, Cambridge, 2019. http://www.iris-kidney.com/pdf/IRIS_Staging_of_CKD_modified_2019.pdf..

  68. Iatta R, Carbonara M, Morea A, Trerotoli P, Benelli G, Nachum-Biala Y, et al. Assessment of the diagnostic performance of serological tests in areas where Leishmania infantum and Leishmania tarentolae occur in sympatry. Parasit Vectors. 2023;16:352.

    Article  PubMed  PubMed Central  Google Scholar 

  69. Calvalido J, Wood GA, Mutsaers AJ, Wood D, Sears W, Woods JP. Comparison of serum cytokine levels between dogs with multicentric lymphoma and healthy dogs. Vet Immunol Immunopathol. 2016;182:106–14.

    Article  CAS  PubMed  Google Scholar 

  70. Sato S, Yanagihara T, Ghazizadeh M, Ishizaki M, Adachi A, Sasaki Y, et al. Correlation of autophagy type in podocytes with histopathological diagnosis of IgA nephropathy. Pathobiol. 2009;76:221–6.

    Article  CAS  PubMed  Google Scholar 

  71. Vogelmann SU, Nelson WJ, Myers BD, Lemley KV. Urinary excretion of viable podocytes in health and renal disease. Am J Physiol Renal Physiol. 2003;285:40–8.

    Article  Google Scholar 

  72. Paparcone R, Fiorentino E, Cappiello S, Gizzarelli M, Gradoni L, Oliva G, et al. Sternal aspiration of bone marrow in dogs: a practical approach for canine leishmaniasis diagnosis and monitoring. J Vet Med. 2013;2013:1–4.

    Article  Google Scholar 

  73. Solano-Gallego L, Rodriguez-Cortes A, Trotta M, Zampieron C, Razia L, Furlanello T, et al. Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis. Vet Parasitol. 2007;147:315–9.

    Article  CAS  PubMed  Google Scholar 

  74. Swets JA. Measuring the accuracy of diagnostic systems. Science. 1988;240:1285–93.

    Article  CAS  PubMed  Google Scholar 

  75. Kim MJ, Tam FWK. Urinary monocyte chemoattractant protein-1 in renal disease. Clin Chim Acta. 2011;412:2022–30.

    Article  CAS  PubMed  Google Scholar 

  76. Zatelli A, Borgarelli M, Santilli R, Bonfanti U, Nigrisoli E, Zanatta R, et al. Glomerular lesions in dogs infected with Leishmania organisms. Am J Vet Res. 2003;64:558–61.

    Article  PubMed  Google Scholar 

  77. Costa FAL, Goto H, Saldanha LCB, Silva SMMS, Sinhorini IL, Silva TC, et al. Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis. Vet Pathol. 2003;40:677–84.

    Article  CAS  PubMed  Google Scholar 

  78. Meneses GC, De Francesco DE, da Silva Junior GB, Bezerra GF, da Rocha TP, de Azevedo IEP, et al. Visceral leishmaniasis-associated nephropathy in hospitalised Brazilian patients: new insights based on kidney injury biomarkers. Trop Med Int Health. 2018;23:1046–57.

    Article  CAS  PubMed  Google Scholar 

  79. Silvestrini P, Zoia A, Planellas M, Roura X, Pastor J, Cerón JJ, et al. Iron status and C-reactive protein in canine leishmaniasis. J Small Anim Pract. 2014;55:95–101.

    Article  CAS  PubMed  Google Scholar 

  80. Liu Y, Xu K, Xiang Y, Ma B, Li H, Li Y, et al. Role of MCP-1 as an inflammatory biomarker in nephropathy. Front Immunol. 2023;14:1303076.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The paper has been sponsored by Elanco Animal Health in the framework of the 18th CVBD® World Forum Symposium. The authors thank Mrs. Alessia Bombasaro, a laboratory technician at San Marco Laboratory, for her technical support, and doctors Matteo Petini and Alissa Mazzei for their help in the graphic creation.

Funding

None.

Author information

Authors and Affiliations

  1. San Marco Veterinary Clinic and Laboratory, Veggiano, Padua, Italy

    Valeria Pantaleo & Tommaso Furlanello

  2. Department of Statistical Sciences, University of Padova, Padua, Italy

    Laura Ventura

  3. Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain

    Valeria Pantaleo & Laia Solano-Gallego

Authors
  1. Valeria Pantaleo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tommaso Furlanello
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laura Ventura
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Laia Solano-Gallego
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.P. and L.S. conceived the study; V.P. and L.S. designed the experiment; L.V. analyzed the data; T.F. participated in the implementation of the study; V.P. wrote the paper; T.F., L.V., and L.S. critically revised the manuscript. All the authors read and approved the final version of the manuscript.

Corresponding author

Correspondence to Laia Solano-Gallego.

Ethics declarations

Ethics approval and consent to participate

All collection procedures were performed solely for the dog’s benefit and for standard diagnostic purposes. Written informed consent was obtained from all owners of the dogs. No anesthesia or euthanasia was required in any part of the study. All procedures complied with the European legislation on the protection of animals used for scientific purposes (Directive 2010/63/EU) and with the ethical requirement of the Italian law (Decreto legislativo 4 March 2014, n. 26). Accordingly, our study did not require authorization or an identification (ID) protocol number.

Consent for publication

All the authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pantaleo, V., Furlanello, T., Ventura, L. et al. Serum and urinary monocyte chemoattractant protein-1 as markers of inflammation and renal damage in dogs with naturally occurring leishmaniosis. Parasites Vectors 17, 366 (2024). https://doi.org/10.1186/s13071-024-06432-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13071-024-06432-0

Keywords

Parasites & Vectors

ISSN: 1756-3305

Contact us