Cavalera et al. Parasites Vectors (2021) 14:525 https://doi.org/10.1186/s13071-021-05030-8 Parasites & Vectors Effect of domperidone (leisguard®) SHORT REPORT Open Access on antibody titers, inflammatory markers and creatinine in dogs with leishmaniosis and chronic kidney disease Maria Alfonsa Cavalera1, Floriana Gernone1, Annamaria Uva1, Paola D’Ippolito2, Xavier Roura3, Saverio Paltrinieri4 and Andrea Zatelli1* Abstract Background: Immunotherapeutic drugs, such as domperidone, have been shown to be promising treatments against canine leishmaniosis (CanL), but limited data are available. The aim of this pilot study (therapeutic, prospec- tive and non-controlled) was to evaluate the effect of domperidone on serum antibody titers of Leishmania infantum, globulins, gamma globulins, acute-phase proteins (e.g. C-reactive protein [CRP]), big endothelin-1 (big ET-1), serum creatinine (SC) and proteinuria in dogs with leishmaniosis affected by chronic kidney disease (CKD). Methods: Dogs were recruited if “exposed” to or “infected” with L. infantum and affected by CKD (IRIS stage 1 [pro- teinuric] or IRIS stage 2–3a [SC < 3.5 mg/dl; proteinuric or non-proteinuric]). After inclusion, an oral suspension of domperidone was administered, and the dogs were followed up for 180 days, with checks at 30, 60, 90 and 180 days after initial treatment. Results: Of the 14 recruited dogs, nine showed a statistically significant reduction in SC (χ2 = 9.1, df = 3, P = 0.028), but not in the urine protein/creatinine ratio (χ2 = 6.43, df = 3, P = 0.092). All dogs showed a significant reduction in antibody titers for L. infantum (χ2 = 9.56, df = 2, P = 0.008), globulins (χ2 = 11.08, df = 3, P = 0.011) and gamma globu- lins (χ2 = 12.38, df = 3, P = 0.006) during the study period. There was also a statistically significant reduction in CRP (χ2 = 16.7,df = 3, P = 0.001), but not in big ET-1 (χ2 = 2.04, df = 3, P = 0.563). Conclusions: This study provides preliminary results on the ability of domperidone to improve SC and reduce anti-L. infantum antibody titers, globulins, gamma globulins and CRP in dogs with leishmaniosis and CKD. Keywords: Antibody titer, Creatinine, CRP, Dog, Domperidone, Gamma globulins, Globulins, Leishmaniosis Canine leishmaniosis (CanL) is a major global sand fly- [4]), clinical disease affects only a limited proportion of borne disease caused by Leishmania infantum, affecting infected dogs [5]. The different outcomes of leishmanio- at least 2.5 million dogs in southwestern Europe [1–3]. sis in canine populations, ranging from infection without Although the high levels of seroprevalence by L. infan- clinicopathological changes to potentially fatal disease, tum in hyperendemic foci (e.g. 28.2% in southern Italy depend mainly on the individual immune response of the affected dog. Indeed, in dogs susceptible to leishmanio- sis, disease progression is due to both a marked humoral *Correspondence: andrea.zatelli@uniba.it immune response and an impaired cellular immune 1 Department of Veterinary Medicine, University of Bari, Valenzano, Italy response against the protozoa [2, 6]. Full list of author information is available at the end of the article © The Author(s) 2021. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cavalera et al. Parasites Vectors (2021) 14:525 Page 2 of 9 The production of anti-Leishmania antibodies does and evidence suggests that the intrarenal ­DA2 receptor in not provide protection against CanL as it leads to hyper- dogs plays a role in the control of renal function [28, 29]. globulinemia and the appearance of soluble circulating Indeed, intrarenal administration of specific ­DA2 recep- immune complexes (CIC) if an adequate antigen/anti- tor antagonist increases glomerular filtration rate (GFR), body ratio, i.e. a moderate prevalence of antigens with renal plasma flow (RPF) and filtration fraction in uni- respect to antibodies, is present [7–10]. Defective clear- nephrectomized dogs [28], while intrarenal D ­ A2 receptor ance of these CIC by scavenging macrophages induces stimulation decreases renal function by hemodynamic their deposition in specific organs, such as in the kidney, mechanisms [29]. resulting in proteinuric glomerulonephritis and, conse- Therefore, based on the considerations outlined above, of domperidone (leisguard®; Ecuphar Italia srl, Milan, quently, tubulo-interstitial lesions [11]. CIC-mediated the primary aim of this study was to evaluate the efficacy renal pathology plays a pivotal role in the prognosis of CanL as chronic kidney disease (CKD) leading to severe Italy) in: (i) maintaining and/or improving renal function renal damage represents the major cause of death in dogs (stable or decreased serum creatinine [SC]) and (ii) main- with leishmaniosis [12]. Therefore, survival time and taining and/or reducing proteinuria (stable or decreased progression of CanL are strongly influenced by an early urinary protein/creatinine ratio [UPC]), in dogs with diagnosis and an appropriate therapeutic approach of leishmaniosis affected by CKD. Moreover, in order to investigated the effect of leisguard® on serum antibody infectious and renal diseases [12]. confirm previous published data [19, 20, 30], we also The first-line treatment protocol for dogs with leish- maniosis and kidney disease includes a leishmanicidal titers for L. infantum and on the concentrations of globu- drug (i.e. meglumine antimoniate at 100 mg/kg twice lins, gamma globulins, C-reactive protein (CRP) and big daily or once daily subcutaneously for 1 month) in com- endothelin-1 (big ET-1) in dogs with leishmaniosis. bination with a leishmaniostatic drug (i.e. allopurinol at This study was a therapeutic, prospective and non- 10 mg/kg orally twice daily or once daily together with controlled field trial conducted in two areas where CanL xanthinuria, for at least 12 months) [13–15]. If this ther- is endemic in southern Italy (i.e. Apulia and Basilicata apeutic regimen is not possible, the second-line treat- regions) [4] from May to November 2018. Privately ment is a combination of miltefosine (2 mg/kg orally owned dogs of any sex, age, weight and breed were once daily for 28 days) and allopurinol [13, 14]. However, recruited for participation in this pilot study if classi- a parasitological cure (i.e. elimination of parasites from fied as “exposed” to (i.e. stage A) or “infected” with (i.e. tissues) with the currently available compounds is rarely stage B) L. infantum according to the Canine Leishma- achieved, and clinical relapses can still appear weeks to niasis Working Group (CLWG) staging system [2] and years after the beginning of treatment [16–18]. if affected by proteinuric CKD (UPC > 0.5) classified as In this scenario, despite the limited data that are cur- IRIS stage 1 up to stage 3a or by non-proteinuric CKD rently available, immunotherapeutic treatments have (UPC ≤ 0.5) classified as IRIS stage 2 up to stage 3a (i.e. shown to be promising against CanL, with the main with SC < 3.5 mg/dl) [31]. objective of re-establishing dog immunity and, there- Dogs were excluded if suspected or known to be fore, promoting parasite reduction and improving clinical affected by: (i) co-infections with other vector-borne signs [19–22]. Indeed, the use of non-specific immune pathogens, such as Ehrlichia canis and Anaplasma modulatory treatments has been reported as potentiat- phagocytophilum; (ii) diseases able to determine progres- ing the immune system of sick dogs to control the infec- sion of CKD and/or to increase the levels of of inflam- tion and to prevent the development of clinical disease in matory markers (e.g. neoplastic, auto-immune and heart uninfected dogs [23–25]. diseases, diabetes mellitus and insipidus, hypo- and Domperidone, an immunotherapy drug, has been hyperadrenocorticism or hyper- and hypothyroidism); shown to be useful for the management of the early (iii) active forms of CanL (i.e. stages C–D according to stages of CanL or for the prevention of clinical disease the CLWG staging system) [2]. All dogs that had been as part of an integrated control program [15, 19, 26]. For administered either leishmanicidal or leishmanio- example, in one study, domperidone was able to induce static treatments in the previous 6 months, as well as clinical improvement in 86% of the dogs affected by leish- those that had also been in therapy for kidney disease maniosis with multiple clinical signs, with serum anti- for < 6 months or whose CKD was not stable, were also body titers decreased by 38% [19]. Indeed, domperidone excluded [32]. enhances the cell-mediated immune response, poten- At inclusion time (T0), each dog was physically exam- tiating the phagocytic and oxidative functions of canine ined, following which blood samples were collected from neutrophils [20]. Domperidone is also a peripherally act- either the cephalic or jugular veins into K3 EDTA tubes ing specific dopamine 2 (­DA2) receptor antagonist [27], (2 ml/tube) to undergo routine hematology and into plain Cavalera et al. Parasites Vectors (2021) 14:525 Page 3 of 9 Fig. 1 Schematic of the measurement time points (days), scheduled procedures and treatment protocol of the study. Abbreviations: CBC, Complete blood count; ELISA, enzyme-linked immunosorbent assay; IFAT, indirect fluorescent antibody test tubes (5 ml/tube) to obtain serum after centrifugation the study, as shown in Fig. 1. At T30, T90 and T180, the (15 min at 1500 g). Urine samples were collected by free dogs were given a complete physical examination, and catch or cystocentesis [33] and put into sterile, evacu- onset or worsening of polyuria and polydipsia (PU/PD) ated, 10-ml collection tubes; these were stored at room (if present), onset or worsening of nausea or vomiting (if temperature (approx. 20 °C [67.6 °F]) and analyzed within present), body condition score (BCS) (based on a scale of 4 h from collection. For each enrolled dog, a complete 1 to 5, with 3 being optimal) and body weight (kg) were blood count (CBC) with reticulocyte count, a complete recorded. At T60, the state of the dogs was followed-up biochemical panel, including APPs (i.e. CRP), big Et-1, by telephone, with the owner asked questions on the gen- electrophoresis and fibrinogen concentration measure- eral clinical condition of the dog, onset or worsening of ments, and a complete urine exam with osmolality and PU/PD (if present), onset or worsening of nausea or vom- UPC were performed. Furthermore, at T0, T90 (follow- iting (if present), BCS evaluation and body weight (kg). up day 90) and T180 (follow-up day 180), the dogs were Statistical differences between SC, UPC, CRP, big tested for anti-L. infantum antibodies using an enzyme- Et-1, globulins, gamma globulins and anti-L. infantum linked immunosorbent assay (ELISA) [34] and for anti-A. antibody titers obtained at T0, T30, T90 and T180 were phagocytophilum (MegaCor Diagnostik, Horbranz, Aus- tested for significance by the Friedman test. A value of tria) and anti-E. canis (Biopronix Agrolabo, Scarmagno, P < 0.05 was considered to be statistically significant. Italy) antibodies by an indirect fluorescent antibody test The statistical analyses were performed using Analyse-it (IFAT) (Fig. 1). (Analyse-it Software, Ltd., Leeds, UK). an oral suspension of domperidone (leisguard®) accord- Immediately after inclusion, dogs were treated with Fourteen dogs were included in this study, as shown in Table 1. Five dogs were intact males and nine were ing to the following dosage and scheme: 1 ml/10 kg females (six intact and three spayed), ranging from 27 to once daily for 4 weeks; 3 months without treatment; 118 months in age and weighing from 3 to 37 kg. Eight 1 ml/10 kg once daily for 4 weeks; finally, 1 month with- dogs were mixed breed, two were English cocker span- out treatment. The owners of the dogs checked prod- iel and one each was beagle, miniature pinscher, Labra- uct intake and the appearance of any side effects [35] dor retriever and boxer. During the clinical trial, one dog throughout the administration period. (#10) was excluded after 27 days from T0 because of a The enrolled dogs were followed up at 30 (T30), 60 surgical procedure due to gastric volvulus, while one dog (T60), 90 (T90) and 180 (T180) days after inclusion in (#5) was withdrawn and euthanized at T90 due to renal Cavalera et al. Parasites Vectors (2021) 14:525 Page 4 of 9 Table 1 Data on the 14 dogs enrolled in the study at T0 Dog ID no. Inclusion (T0) IRIS stage/NP, Creatinine (mg/dl) RI: UPCb Leishmania infantum Globulins (g/dl) RI: CRP (mg/dl) RI: Big ET-1 (pg/ml) BP, ­Pb 0.70–1.40 ELISA (%)c 2.8–3.9 0.01–0.45 1a 1/BP 1.07 0.21 Low (18.3) 4.1 0.16 2.4 2a 1/P 0.84 1.27 Medium (20.8) 3.6 0.05 2.1 3 1/P 0.74 4.5 High (33.9) 3.4 0.08 3.0 4 1/P 0.95 0.54 Low (14.7) 2.9 0.69 2.0 5a 1/P 0.55 1.69 Low (14.8) 3.1 0.03 NA 6 1/BP 1.12 0.23 High (32.0) 4.5 0.09 1.8 7 1/P 1.00 1.21 Medium (22.3) 3.8 0.29 1.6 8 2/P 2.37 1.74 High (30.5) 4.0 0.24 NA 9 1/BP 0.60 0.41 Medium (24.7) 4.0 0.41 1.8 10a 1/P 1.20 2.07 Low (17.5) 3.7 0.02 2.0 11 1/BP 1.12 0.21 Low (18.1) 4.3 0.19 1.9 12 1/BP 1.22 0.33 High (31.2) 4.5 0.12 1.8 13 1/P 1.10 0.60 Low (18.6) 4.1 0.32 NA 14a 1/BP 0.60 0.41 High (29.5) 4.0 0.41 2.1 All dogs were receiving treatment for chronic kidney disease for > 6 months Big ET-1 Big endothelin-1, CRP C-reactive protein, ELISA enzyme-linked immuosorbent assay, NA not applicable, RI reference interval, T0 inclusion time point, UPC urinary protein/creatinine ratio a Dogs excluded from the study at different time points b IRIS stages: 1–4. NP, Non-proteinuric (UPC < 0.20); BP, borderline proteinuric (UPC = 0.21–0.50); P, proteinuric (UPC > 0.50) [31] c Low 13–20%; medium 20.1–25%; high > 25% amyloidosis diagnosed by increased UPC and histologic (χ2 = 2.04, df = 3, P = 0.563), when values at T0, T30, T90 findings from the post-mortem renal specimen biopsy and T180 were compared (Fig. 2). No further alterations (i.e. positive staining for Congo Red) (Table 1). in the parameters included in the biochemical panel were Three dogs were excluded because of recurrence of recorded (e.g. electrolytes) during the study period. In active leishmaniosis (two dogs at T90 [#1, #14] and one addition, all enrolled dogs had an inactive urinary sedi- dog [#2] at T180), diagnosed by clinical signs and/or ment and urine specific gravity (USG) within normal lim- pathological alterations, such as increased CRP and/or its (1015–1045). globulin values, hypalbuminemia and reduced albumin/ All nine dogs that completed the study tested negative globulin ratio, as well as direct identification of amas- to anti-A. phagocytophilum and anti-E. canis antibodies tigotes in lymph node (n = 1) or bone marrow (n = 2) by IFAT at T0, T90 and T180. No side effects related to aspirates and increased anti-L. infantum antibody titer domperidone administration were reported [34]. (Table 2). cacy of domperidone (leisguard®) in improving renal This pilot study provides preliminary data on the effi- All nine dogs that completed the entire pilot study showed a statistically significant reduction in SC function (SC) in dogs with leishmaniosis affected by (χ2 = 9.1, df = 3, P = 0.028) (Fig. 2), but not in UPC CKD. The data from the present study also confirm previ- (χ2 = 6.43, df = 3, P = 0.092). Furthermore, even the three ous published results on the ability of this drug to signifi- dogs that developed a recurrence of leishmaniosis during cantly reduce anti-L. infantum antibody titers [19] while the trial course and were withdrawn at T90 (n = 2) and showing for the first time a potential effect in improving at T180 (n = 1) maintained a stable serum creatinine level clinical and laboratory values for globulins, gamma glob- (Table 2). ulins or other inflammatory markers, such as CRP. During the study period, there was a significant High levels of antibodies are associated with a high reduction in all dogs of antibody titers for L. infantum parasitic burden [36]. Consequently, the reduction of (χ2 = 9.56, df = 2, P = 0.008), globulins (χ2 = 11.08, df = 3, antibody titers is related to the ability of domperidone P = 0.011) and gamma globulins (χ2 = 12.38, df = 3, to indirectly cause a regression of antigenic stimulation P = 0.006) (Fig. 2). The Friedman test showed a statis- by L. infantum. Indeed, domperidone activates dogs’ tically significant reduction in CRP during the study cell-mediated immune response by increasing the levels period (χ2 = 16.7, df = 3, P = 0.001), but not in big ET-1 of prolactin in blood, with prolactin considered to be a Cavalera et al. Parasites Vectors (2021) 14:525 Table 2 Comparison of creatinine, urinary protein/creatinine ratio, anti-L. infantum antibody titers, globulins, C-reactive protein and big endothelin-1 values of the three dogs which developed a recurrence of leishmaniasis during the trial course and were withdrawn at T90 (n = 2) and at T180 (n = 1) Dog ID no. T0 T90 (dog #1, #14)/T180 (dog #2) a Creatinine UPC L. infantum ELISA (%) Globulins (g/dl) CRP (mg/dl) Big ET-1 (pg/ml) Creatinine UPC L. infantum ELISA (%)a Globulins (g/dl) CRP (mg/dl) Big ET-1 (pg/ml) (mg/dl) (mg/dl) 1 1.07 0.21 Low (18.3) 4.1 0.16 2.4 0.93 2.10 High (30.7) 8.6 7.04 2.8 2 0.84 1.27 Medium (20.8) 3.6 0.05 2.4 0.69 2.16 High (34.3) 5.4 0.15 2.4 14 0.60 0.41 High (29.5) 4.0 0.41 2.1 0.60 1.61 Medium (23.8) 5.1 1.16 3.6 T90, T180 90 and 189 days after inclusion day/start of initial treatment a Low 13–20%; medium 20.1–25%; high > 25% Page 5 of 9 Cavalera et al. Parasites Vectors (2021) 14:525 Page 6 of 9 Fig. 2 Box-plot graph of creatinine, C-reactive protein (CRP), globulins and anti-L. infantum antibody titers obtained at T0, T30, T90 and T180. Values were tested for significance by the Friedman test proinflammatory lymphocyte-derived cytokine [19, 37, titers for L. infantum (χ2 = 9.56, df = 2, P = 0.008), globu- 38]. A prolactin-mediated stimulation of the Th1 immune lins (χ2 = 11.08, df = 3, P = 0.011)and gamma globulins response causes release of interleukin (IL)-2, IL-12, inter- (χ2 = 12.38, df = 3, P = 0.006), could not be excluded. feron (INF)-γ and tumor necrosis factor (TNF)-α, which Indeed, the major cause of glomerular pathology in CanL leads to activation of natural killer cells and macrophages is represented by the deposition of preformed CIC at followed by a decrease in CD4 + Th2 subsets and TNF-β different levels of the glomerular unit, triggering local- [39–41]. According to this scenario, domperidone could ized inflammation [12]. Domperidone might reduce the promote L. infantum load control and prevent progres- formation of CIC due to the decrease of antigenic stim- sion of the disease. Interestingly, the reduction in anti- ulation, obtained by activating the phagocytic polymor- body titer in the enrolled dogs shown by the ELISA phonuclear cells involved in the innate immune response occurred not only in the presence of a initial antibody and the reduction of antibody production [19, 20]. There- titer (high, medium or low) but also throughout the sand fore, SC reduction might be associated to a reduction of fly season in the study area (i.e. from May to November; CIC, as previously described, where SC level was signifi- [42]) during which time antibody levels generally tend to cantly correlated to CIC levels during CanL [9, 45]. rise [43]. It is noteworthy that the degree of reduction in Although outside the main aims of the study, attention humoral response could be affected by the response of should be paid to the three dogs that developed a recur- the individual dog [44]. rence of leishmaniosis during the trial course, presenting The study population showed a statistically signifi- an increase in anti-L. infantum antibody titers, globulins, cant reduction in SC (χ2 = 9.1, df = 3, P = 0.028). The CRP and UPC (Table 2). All three dogs maintained a sta- renal effects of domperidone in leishmaniotic dogs with ble SC level despite clear evidence of a relapse of leish- CKD can be considered to be a result of either a “direct” maniosis and in contrast with previously published data or “indirect” intrarenal action. According to [28], the that demonstrated a strict relationship between CIC and “direct” action can be associated to the ability of spe- SC level [9, 45], thus making also a direct intrarenal effect cific ­DA2 receptor antagonists being able to increase of domperidone more likely. GFR, RPF and the filtration fraction, thereby potentially On the other hand, although it is possible to detect a promoting renal filtration and the maintenance of ade- free light-chain proteinuria in absence of structural or quate SC levels. An “indirect” action of domperidone functional renal derangement in dogs with leishmanio- on the kidney due to the improvement of CanL-related sis [46], values of proteinuria usually mirror the extent parameters, such as a significant reduction in antibody of the glomerular kidney damage due to the deposition Cavalera et al. Parasites Vectors (2021) 14:525 Page 7 of 9 of CIC [11, 12]. Therefore, a treatment which would be the clinical application of domperidone (leisguard®) for the able to improve renal perfusion, such as domperidone management of CanL. treatment, would be unlikely to have a direct impact on In conclusion, this pilot study provides preliminary proteinuria. Indeed, none of the enrolled dogs showed results on the ability of domperidone to improve kidney a statistically significant reduction in UPC. Also to be function by reducing anti-L. infantum antibody titers, glob- considered is the possibility that domperidone, by ame- ulins, gamma globulins and CRP in dogs with leishmanio- liorating RPF, might determine intraglomerular hyper- sis affected by CKD. However, further studies are necessary tension, thus conditioning the grade of proteinuria [47]. to test the efficacy of domperidone in improving the kidney In this study globulins, gamma globulins and CRP function of dogs with leishmaniosis in advanced stages of showed a significant decrease over time. Although pre- CKD and to elucidate the effect of this drug in L. infantum- treatment CRP values were within normal limits in all negative dogs affected by renal insufficiency. enrolled dogs (Table 1), the significant reduction in this APP suggests that some activation of inflamma- Abbreviations tory/immune responses occurred secondary to expo- APPs: Acute-phase proteins; BCS: Body condition score; big ET-1: Big endothe- sure to the parasite. At the same time, the CRP values lin-1; CanL: Canine leishmaniosis; CBC: Complete blood count; CIC: Circulating at T0 support the hypothesis that the enrolled dogs did immune complexes; CKD: Chronic kidney disease; CRP: C-reactive protein; DA2: Dopamine 2; GFR: Glomerular filtration rate; IFN: Interferon; IL: Interleukin; not have an active infection and that spreading of the PU/PD: Polyuria and polydipsia; RPF: Renal plasma flow; SC: Serum creatinine; parasite typically occurs only in sick dogs [2, 44], as TFN: Tumor necrosis factor; UPC: Urinary protein/creatinine ratio; USG: Urine occurred in the three dogs withdrawn from the study specific gravity. (Table 2). This result regarding CRP substantially con- Authors’ contributions firms findings from previous studies on the short-term AZ and PDI conceived and designed the project. AZ, PDI and AU performed effect on domperidone on CRP concentration: after a the sampling. MAC, SP and AZ did the data analysis. MAC wrote the first draft of the manuscript. MAC, FG, AU, PDI, XR, SP and AZ revised the manuscript. AZ transient increase in the first days of treatment consist- supervised the project. All authors read and approved the final manuscript. ent with a domperidone-induced increased metabo- lism of phagocytes, there was a trend to a decreased Funding This research and the APC were funded by Ecuphar Italia srl, viale CRP concentration towards the end of the first month Francesco Restelli n 3/7 piano 1, 20124 Milan (Italy), grant number of treatment [19, 44]. IT0189/2018-PRVPRO-IP00. The lack of a significant decrease of Big-ET1 during the Availability of data and materials study period likely depends on the very low level of this The datasets generated and/or analyzed during the current study are available analyte recorded at T0, which hampered the possibility from the corresponding author on reasonable request. to observe further decreases during the follow-up. Big- ET1 is known to increase in response to hypertension Declarations associated with advanced stages of CKD [48]. Therefore, Ethics approval and consent to participate the very low level of this APP can be related to the fact A written informed consent form was signed by each owner before the that enrolled dogs, despite the possible activation of the enrollment of their dog in the study. The administered product has been used inflammatory or immune responses mentioned above, according to the manufacturer’s instructions as stated in the package leaflet. were not in an advanced kidney disease stage. Consent for publication The main limitations of the present study are the absence Not applicable. of a CanL-positive control group not treated with domperi- Competing interests done and the small sample size due to the application of The sponsors had no role in the design, execution, interpretation, or writing strict inclusion criteria. The pilot nature of the study and of the study. its realistic constraints (such as budget limits) also affected Author details the number of dogs that could be enrolled. Moreover, 1 Department of Veterinary Medicine, University of Bari, Valenzano, Italy. dogs enrolled in the study did not undergo renal biopsy; 2 Veterinary Diagnostic Lab ACV Triggiano, Triggiano, Italy. 3 Hospital Clínic thus, there was no complete characterization of the renal Veterinari, Universitat Autònoma de Barcelona, Bellaterra, Spain. 4 Department of Veterinary Medicine, University of Milan, Milan, Italy. damage or a final histopathological classification accord- ing to current guidelines [2, 31]. However, the character- Received: 23 July 2021 Accepted: 21 September 2021 istics of the enrolled animals, such as clinical stability for CKD, made a renal biopsy unnecessary according to cur- rent indications [49, 50]. 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Consensus guidelines for immunosuppressive treatment of dogs with glomerular disease absent a pathologic diagnosis. J Vet Intern Med. 2013;27(Suppl. 1):S55–9. Ready to submit your research ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations • maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions