Hindawi Veterinary Medicine International Volume 2019, Article ID 6073624, 5 pages https://doi.org/10.1155/2019/6073624 Research Article Prevalence of Proteinuria in Owned Dogs from Italy: A Multicentric Study M. Gizzarelli,1 X. Roura,2 P. Scarpa ,3 P. D*Ippolito,4 V. Foglia Manzillo ,1 G. Oliva,1 A. Tarducci,5 A. Borrelli,5 G. Melis,6 F. Quintavalla,6 A. Uva,7 A. Guarraci,8 and A. Zatelli4 1 Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy 2 Hospital Clı́nic Veterinari, Universitat Autònoma de Barcelona, Bellaterra, Spain 3 Department of Veterinary Medicine, University of Milan, Milan, Italy 4 R&D Department, Medical Consultancy Services, Ta’Xbiex, Malta 5 Department of Veterinary Sciences, University of Turin, Grugliasco, Italy 6 Department of Veterinary Science, University of Parma, Parma, Italy 7 Ospedale Veterinario Pingry, Bari, Italy 8 Clinica Veterinaria San Lorenzo, Palermo, Italy Correspondence should be addressed to V. Foglia Manzillo; valentina.foglia@unina.it Received 10 December 2018; Revised 2 March 2019; Accepted 6 March 2019; Published 19 March 2019 Academic Editor: Remo Lobetti Copyright © 2019 M. Gizzarelli et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Even though proteinuria is related to different causes, when it is persistent and associated with inactive urinary sediment, it is primarily due to kidney disease. Early detection of proteinuria allows us to identify several pathological conditions. The aim of the study was screening a canine population not known as being proteinuric, by the urinary dipstick. The study was carried out in seven Italian veterinary clinics during a period of six weeks. Dogs were enrolled with no restriction of sex or age. Females in estrus, dogs with signs of genitourinary diseases, or those previously diagnosed with proteinuric nephropathy were excluded. Dogs were considered “nonproteinuric” (NP) in case of negative dipstick test or “suspected proteinuric” (SP), if positive at the dipstick. When possible, proteinuria was confirmed by UPC ratio. A total of 1156 dogs were evaluated: 414 were from northern Italy and 742 from southern Italy. Based on dipstick test, 655 (56.6%) dogs were NP, while 501 (43.3%) were SP. Among the NP dogs 225 out of 414 (54.3%) were in northern Italy and 430 of 742 (57.9%) in southern Italy. One hundred eighty-nine of 414 (45.7%) SP dogs were identified in northern Italy and 312 of 742 (42.1%) in southern Italy. No statistical difference was found between the North and the South of Italy. UPC was available in 412 out of 501 SP samples: proteinuria was confirmed in 263 (63.86%) samples. Results from our study showed a high percentage of suspected proteinuric dogs, apparently not affected by renal diseases, together with the absence of statistically significant differences based on geographical area. 1. Introduction due to renal causes compared to nonproteinuric dogs [2– 7]. Moreover, the response to the antiproteinuric therapy Proteinuria is defined as the presence of an excessive amount influences the progression of the disease: the possibility of of proteins in the urine, or of abnormal proteins that are instituting an antiproteinuric therapy, associated with the not normally present [1–4]. Over the last years, canine pro- early diagnosis of proteinuric nephropathy, has proved to be teinuria has assumed a position of prominence and growing effective in reducing the risk of mortality due to renal causes interest enough to be now considered not only a marker of in several natural models of the disease [3, 4, 7]. nephropathy, but also a prognostic factor [2–5]. Proteinuric Causes of proteinuria of renal origin include either dogs, for the same IRIS stage, show a different progression genetic or infectious diseases, other than all diseases leading of the nephropathy depending on the amount of protein lost to a chronic stimulation of the immune system and immune- in the urine and they have an increased risk of mortality mediated disorders in general. Many of the underlying 2 Veterinary Medicine International conditions of the proteinuric nephropathy are not identified healthy canine population revealed a high prevalence of renal or are identified belatedly and this makes the management disease, thus leading the authors to propose the screening of of the renal disease difficult; furthermore, the lack of easily all senior dogs, even those considered healthy, to allow the identifiable clinical signs often makes the diagnosis late. diagnosis of renal disease otherwise not identifiable at the Being the presence of proteinuria a marker of nephropa- clinical visit [16]. In Italy, data regarding the prevalence of thy as well as an important prognostic factor in dogs affected proteinuria in dogs are not available. by kidney disease, screening of the population at risk can Main aim of the present study was screening a canine assume a role of particular importance as most proteinuric population, living in Italy and not known as being pro- dogs are not identified at the early stages of the disease teinuric, by the urinary dipstick. When available, the UPC for the lack of clinical signs [3, 4, 7, 8]. In order to carry values obtained from the urine of dogs positive at the it out successfully, the screening of the canine population dipstick were evaluated to confirm or exclude protein- not only requires the identification of dogs at risk of the uria and to determine its prevalence in the screened disease, but also it is necessary that the tests chosen are easy population. to use, of low costs, and performed on samples collected with no invasive procedures. In dogs, the urinary dipstick for the identification of nonproteinuric patients has been 2. Material and Methods proposed [2, 9, 10]. Dogs whose urine is negative at the The study was conducted in Italy over a period of 6 weeks. dipstick can be considered nonproteinuric, while all samples Seven clinics participated and enrolled the dogs: 4 were with positive results should undergo the more sensitive and located in northern Italy (Milan, Turin, Parma, and Genoa) specific urinary protein to creatinine ratio (UPC) [10]. Urine and 3 in the south of Italy (Naples, Bari, and Palermo). All collection by spontaneous micturition (free catch) has also dogs not known as being proteinuric, regardless of the age or been validated in dogs [8, 11]. Positive results at dipstick gender, were included. Females in oestrus and dogs known will require us to rule out any underlying inflammatory or suspected to be affected by diseases of the genitourinary process (through the urinary sediment evaluation), which tract and/or from any diseases determining proteinuria were will finally allow us to identify false positive results [8, 11]. excluded. Dogs in treatment with drugs influencing the level Prerenal causes of proteinuria determining positive results at of proteinuria were also excluded from the study. All owners the dipstick are more commonly related to easily identifiable were informed of the aims of the study and no dog underwent clinical pictures, such as fever and seizures. invasive urine sampling (cystocentesis), if not required by The causal relation between some diseases determining a the practitioner for a diagnostic work-up not related to the chronic stimulation of the immune system and glomerular study. disease causing proteinuria in dogs is suspected. Chronic disease of the skin, as well as periodontal disease, may in fact represent aetiologically important factors which could 2.1. Urine Collection and Storage of the Urine Samples. All urine samples were collected by free catch or cysto- considerably widen the number of dogs at risk of proteinuria [12, 13]. centesis [8, 11]. The urine samples were then stored in a Since the presence of proteinuria can be associated dedicated container, kept at room temperature, and exam- with genetic disease or else determined by several diseases ined with the dipstick (Mindray) within one hour from collection. causing glomerular damage on immune-mediated basis and the proteinuric renal disease is difficult to be identified When possible, urinary sediment of samples positive to (especially in its early stages) if not through laboratory tests, the dipstick was evaluated and, if inactive, the UPC was then determined. All urine samples undergoing the UPC the identification and the screening of the population at determination were immediately centrifuged at 1500 rpm for risk play a key role for the early diagnosis of proteinuric nephropathy [3, 4, 6, 14]. Breeds at risk of renal disease for 10 minutes and the supernatant was then separated and put genetic reasons should be screened early (within 6 months of into sterile tubes at −20∘ C, if the UPC was not performed age), and all dogs of medium-senior age would deserve a renal within 6 hours from collection [17]. To measure the UPC function evaluation together with a urinalysis at least every 12 ratio, each clinic applied a standardized technique among the months [15]. ones already validated for the purpose. The canine population at high risk of proteinuric nephropathy, for example, because of infectious diseases, 2.2. Classification of the Results of the Urinary Test. Based on should be screened every 6 months. the dipstick results, dogs were considered either [10] Given the multiple conditions able to determine pro- teinuria in dogs, epidemiological data represent a key factor (i) nonproteinuric (NP): dogs with negative result at in setting up a screening of the population; based on high dipstick, or prevalence of the disease, screening could be addressed not (ii) suspected proteinuric (SP): dogs in which dipstick only to breeds at risk for developing proteinuria or categories gave any positive results (any colorimetric changes). potentially at risk such as dogs affected by vector-borne diseases (e.g., leishmaniosis, ehrlichiosis, anaplasmosis, or When the UPC determination was available, dogs classi- borreliosis), but also to the entire canine population. More- fied as SP were further divided into three different substages over, epidemiological studies carried out in Belgium on a in accordance with the IRIS staging system [6]: Veterinary Medicine International 3 Table 1: Dogs classified as non- or suspected proteinuric on the basis of the screening carried out with reactive strip for urine. In this table the absolute numbers for geographical distribution and the relative percentages to facilitate the comparison of results for the different geographical areas are reported. Number of dogs Nonproteinuric dogs Suspected proteinuric dogs Northern Italy 414 225/414 (54.3%) 189/414 (45.7%) Southern Italy 742 430/742 (57.9%) 312/742 (42.1%) Italy 1156 655/1156 (56.7%) 501/1156 (43.3%) (i) Nonproteinuric (NP): dogs with UPC less than 0.2 results agree with a previous article that revealed a high preva- (ii) Borderline proteinuric (BP): dogs with UPC between lence of proteinuria in dogs of advanced age and geriatrics 0.2 and 0.5 [16]. An interesting aspect of this study resided in the char- acteristics of the canine population subjected to screening, (iii) Proteinuric (P): dogs with UPC above 0.5. because all dogs were considered to be apparently clinically healthy. The authors, confident that often the clinical signs 2.3. Statistical Analysis. The statistical analysis was per- of renal disease are subtle and difficult to identify, concluded formed using MedCalc software (Frank Shoonjans, V.7.2.1.0) by recommending measurement of proteinuria as part of and SPSS, version 13.0 for Windows. Differences in preva- geriatric health screening [16]. The same recommendation lence between different sites were assessed by the two-sided had already been provided by the American Animal Hospital chi-square test t. A P value <0.05 was considered statistically Association (AAHA) for dogs of mature age and seniors [18]. significant. The AAHA suggestions lead us to lower the age in which to begin the screening of the canine population for kidney 3. Results disease compared to what was suggested by Marynissen and colleagues [8]. AAHA guidelines, in fact, consider these During the period of the study, a total of 1156 dogs were dogs “mature,” which have reached 50% of the expected screened for proteinuria, with no homogenous distribution lifespan. Similarly, the International Renal Interest Society among the clinics: 414 dogs were screened in northern Italy (IRIS) recommends the screening for renal disease once a and 742 in southern Italy. Based on dipstick results 655 year for dogs of advanced age, even if they are apparently (56.6%) dogs were NP, while the remaining 501 (43.3%) were healthy [6]. The IRIS also recommends a renal screening SP. Among the NP dogs 225 out of 414 (54.3%) were in every 6 months for all dogs at risk of renal disease including northern Italy and 430 of 742 (57.9%) in southern Italy. One in this category dogs affected by diseases known to determine hundred eighty-nine of 414 (45.7%) SP dogs were identified renal damage [6]. Despite the fact that most of the vector- in northern Italy and 312 of 742 (42.1%) in southern Italy. The borne disease are to be encountered as causing renal damage, results of the screening with the urinary dipstick are reported a role of primary importance (at least for the prevalence in the in Table 1, where the screened population was subdivided general canine population) could be attributed to conditions based on the geographical distribution and the test results as potentially capable of determining a chronic stimulation of NP or SP. the immune system, such as periodontal disease and certain A comparison was made between the two distributions skin diseases [12, 13]. relating to the percentage of negativity and positivity to the Suggestions provided by AAHA, IRIS, and other authors screening test performed by the dipstick and no statistical must be taken in either the social realities or the geographical difference was found between the North and South of Italy. locations where dogs live, as indications of screening from a UPC was available in 412 out of 501 SP samples at dipstick. certain age and over may be not applicable in all contexts, Based on the IRIS staging system (IRIS, 2015), 128/412 (31.0%) because of the presence of diseases potentially predisposing were in substage BP, 135/412 (32.8%) were in substage P, and the onset of renal damage [3, 4, 6, 14, 16, 18]. The suggestion 149/412 (36.2%) were in substage NP. In total, UPC confirmed of screening the entire canine population to promote an early proteinuria of any degree in 263 (63.86%) of 412 urine samples diagnosis of proteinuric renal disease implies the assessment analysed. of three elements: (i) the possibility to modify the prognosis Although complete data were available only for 391 dogs thanks to an early diagnosis and an effective treatment, (ii) from 2 veterinary clinics located in northern Italy and one the possibility to perform the screening by a noninvasive in the south, there were no statistically significant differences sampling and a low cost exam, and (iii) high prevalence of between dogs younger or older than 6 years of age (p>0.05). the disease. Proteinuric nephropathy is a disease usually at slow 4. Discussion progression (thus, a long-term one) and high incidence (par- ticularly, in certain geographical areas endemic, for example, Based on dipstick results, the present study was suggestive of for vector-borne diseases) and for the high prevalence of low prevalence of nonproteinuric dogs and high prevalence other concomitant illnesses, such as, for example, chronic of suspected proteinuric ones from an apparently not-renal cutaneous and periodontal disease. When epidemiologically affected population (not known to be nephropathic). These studied, both slow progression and high incidence are the 4 Veterinary Medicine International two main characteristics of diseases at high prevalence. In method. A recent study by Rossi G. and colleagues showed Spain, a prevalence of proteinuric renal disease ranging from how analytic variability and method-dependent differences 30.2% to 52.7% has been reported in the canine leishmaniosis could affect substaging of patients according to the Interna- population [19]. The high prevalence of proteinuria reported tional Renal Interest Society (IRIS), particularly regarding by the Spanish authors can obviously be related to leish- the values of UPC close to the cut-offs [21]. It is also worth maniosis, which is well known to cause glomerular damage mentioning that the same author also demonstrated the same and consequently proteinuria. Italy, as a country with dif- method but different reagents can further influence a correct ferent geographical areas endemic for vector-borne diseases substaging of the patient. [22]. These two potential biases (e.g., ehrlichiosis, anaplasmosis, and leishmaniosis), could resulting from the determination of the UPC should in any be epidemiologically comparable to Spain. Results of our case be considered as belonging to the clinical practice. The screening allowed us to classify only 56.6% of the population lack of statistically significant difference in the prevalence of of dogs as NP, which is a very low prevalence considering the proteinuria among the structures participating in the study fact that the population tested was supposed to be healthy. could be consequent to the repetition of bias over the involved Furthermore, our data confirmed extremely high prevalence structures. Finally, a further limitation is represented by of proteinuria with 43.3% of the tested dogs as SP at the the selection of the population. For enrolment, the canine urinary dipstick, with 24.6% confirmed to be P or BP at the population had to be considered as apparently not affected UPC determination. However, this study has also revealed by diseases capable of determining proteinuria. Thus, the no difference in the prevalence of nonproteinuric dogs in capacity of the veterinary surgeon had a fundamental role in northern Italy compared to the south of Italy. This is not in the selection of cases, as no laboratory examination could be line with what we had expected, the areas of southern Italy used to further classify the dogs. Although there may be some being characterized by a higher prevalence of vector-borne bias, it is also true that these are the same encountered in daily diseases (potentially determining renal damage) than the practice. Moreover, considering the prevalence of proteinuria areas of northern Italy [20]. Many could be the reasons for the based on age, a limitation can be represented by the arbitrary data obtained, which show no statistical difference between subdivision based on age, which does not consider size and/or the prevalence of proteinuria in endemic and nonendemic breed of dog. In fact, a dog belonging to a breed with an areas for vector-borne diseases. First of all, it should be average lifespan of 9 years has passed 50% of the expected mentioned that many dog owners who live in the north of lifetime at 6 years, while a dog with an expected lifespan of Italy used to move to the coastal areas and the south of 15 years has got only 40% of the expected lifetime at 6 years Italy over summer, where endemic diseases transmitted by [18]. Of course, the risk of kidney damage also increases in vectors are widespread. Despite the use of vector repellents relation to age and since no data about either breed or size and ectoparasiticides, there is the likelihood that dogs staying of the population studied is available, it was not possible to for even short periods of time in endemic areas contract evaluate a possible correlation between positivity/negativity infections that hesitate in seroconversion and subsequent with the urinary dipstick and the biological age of the dogs. production of circulating immunocomplexes, with final renal It is possible that in some clinics dogs of advanced biological damage. The relevance of the movement of dogs via the rescue age have prevailed on those of younger biological age. The fact associations, which have become particularly numerous in that among the three clinics in which complete data about recent years and frequently characterized by the movement of the age of the dogs subjected to screening were available dogs from southern Italy to the north, should also be stressed. significant differences did not come out may suggest that Coexisting diseases potentially causing renal damage could dogs of different breeds and/or sizes as well as different ages represent a further element. Undoubtedly, the risk of peri- (biological also) may have been well represented. Last but odontal disease in dogs does not recognize geographical not least we do not evaluate the serological positivity for differences (or, at least, we are not aware of this so far), and vector-borne disease of dogs, which underwent the screening other diseases may have contributed to determine a uniform for proteinuria, and this makes it impossible to assume a prevalence in the proteinuria distribution on Italian territory. correlation between infectious disease and proteinuria itself A final interesting data is the lack of statistically significant in the population studied and the possible enrollment of difference based on age (p>0.05). Even if it is limited to only subclinical patients. three clinics of the seven enrolling the dogs to be screened, there was in the canine population ages less than or equal to/over 6 years. 5. Conclusions This study has several limitations. First of all, the screen- ing of the canine population has been carried out using the The results of this study are suggestive of a low prevalence urinary dipstick; a test with greater sensitivity and specificity of nonproteinuric dogs with a high prevalence of suspected could have provided more accurate results; however it is proteinuric dogs, together with the absence of statistically undoubted that a negative result at the dipstick is reliable significant differences based on geographical area, for pro- enough to identify dogs to be considered with high probabil- teinuria of dogs living in Italy. These results also suggest ity of NP [9, 10]. Another important limitation is represented proposing periodical screening, possibly annually. 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