Dimensions and skeletopy of kidneys in two populations of Cerdocyon thous (Linnaeus, 1766)

Article history Cerdocyon thous is the South American canid with great geographic distribution. To the south of Ecuador, two isolated populations have been identified, living in different average temperatures and food availability. The objective was to measure the length, width, thickness and volume of the kidneys, length of renal vessels, and verify the renal skeletopy in two populations of C. thous. Kidneys and renal vessels were measured from 34 cadavers collected on highways in the Brazilian territory. From the Atlantic Forest biome (latitude 22o), 14 specimens (seven males and seven female) were analyzed, and from the Pampa biome (latitude 29o), 20 specimens (eight males and twelve female). On average, in the right antimere the kidneys measured 49.9  25.2  24.4 mm, had a volume of 16.5 cm3, the renal artery measured 21.3 mm and the renal vein 19.4 mm. In the left antimere, the kidneys measured 49.3  24.4  22.8 mm, with a volume of 14.6 cm3 and the artery and vein measured 21.0 mm and 28.4 mm, respectively. The right kidney was always cranial and predominantly positioned ventrally to vertebrae L1–L3, while the left one was positioned ventrally to vertebrae L2–L4. There was no difference in the comparison between sexes or antimeres. Most renal dimensions were significantly higher in the specimens from the Pampa biome, possibly due to the body size and type of diet. This result warns of interpreting diagnoses that depend on the evaluation of the normal renal dimensions in this species. Received 23 March 2020 Accepted 28 April 2020


INTRODUCTION
Cerdocyon thous, also known as "crab-eating fox," is the wild canid with the largest distribution area in the South American continent. With great capacity to adapt to habitats in the Neotropics, C. thous inhabits areas of closed or open vegetation (COURTENAY; MAFFEI, 2004;HUNTER, 2011;KASPER et al., 2014). Two disjointed dispersion areas are recognized in South America: one to the north and the other to the south of Ecuador. To the north of Ecuador, the dispersion area is distributed in Colombia, Venezuela, Guyana and Suriname; to the south of Ecuador, it is found in Brazil, Paraguay, Argentina, Uruguay and Bolivia (COURTENAY; MAFFEI, 2004). C. thous has a body mass of 5-9 kg and can measure up to 1.2 m from the snout to tail (KASPER et al., 2014). An omnivore, it eats fruits, insects, crustaceans, small vertebrates and eggs (HUNTER, 2011). Although its conservation is not threatened, C. thous suffers pressure from hunting by rural producers to obtain the skin, is run over by cars, and is infected with diseases transmitted by domestic dogs (HUNTER, 2011;KASPER et al., 2014).
The kidneys are paired, sublumbar retroperitoneal organs located ventrally to the hipaxial musculature and laterally to the aorta artery and caudal vena cava. Each kidney has two surfaces (dorsal and ventral), two poles (cranial and caudal), and two margins (lateral and medial) (NICKEL; SCHUMMER; SEIFERLE, 1979). The medial surface contains the renal hilum that defines a space, the renal sinus, which contains the ureter, blood vessels, lymphatic vessels, and nerves (NICKEL; SCHUMMER; SEIFERLE, 1979). The renal artery has a dorsal position in relation to the renal vein. In dogs, renal vein duplicity is more common than the renal artery (EVANS; LAHUNTA, 2013).
The high occurrence of C. thous in nature and its high frequency in zoos and private collections reflects in the hundreds of annual veterinary services to individuals of this species (COURTENAY; MAFFEI, 2004;SILVA et al., 2014). Consequently, several studies report diagnoses of diseases (FREDO et al., 2015;RIBEIRO;VEROCAI;TAVARES, 2009), biochemical test values (MATTOSO et al., 2015SILVA;NOGUEIRA;SANTANA, 1993), ultrasound evaluation (SILVA et al., 2014, and surgical procedures (PICCOLI et al., 2017) to the kidneys of C. thous.
Knowledge of the renal dimensions is fundamental for diagnosis in nephrology and urology (BARR; HOLT; GIBBS, 1990;KONDE et al., 1984). Although there are reports on kidney measurements in domestic LAHUNTA, 2013;NICKEL et al., 1979;STOCCO et al., 2016) and wild carnivores (EVANS; SOUZA et al., 2018), no studies report the normal dimensions of the kidneys of C. thous. In anatomical terms, one study describes the sectoral intrarenal arterial branching in one female specimen of C. thous (MENEZES et al., 2011) and another reports on one case of duplicity of the left renal artery (PEÇANHA et al., 2020). In addition, the C. thous size has significant intra-specific variation, tending to be larger in populations farther south of the South American continent (BUBADUÉ et al., 2016;MARTINEZ et al., 2013). Therefore, it seems plausible to assume that the normal average dimensions of the kidneys may differ in different populations of the same species.
The aim of this study was to establish kidney and renal vessel measurements and renal skeletopy in individuals from two isolated populations of C. thous, to subsidize procedures in wildlife medicine, and to clarify if the size of the kidneys varies depending on the population's habitat.

MATERIAL AND METHODS
Thirty-four adult specimens of C. thous were collected dead on highways of the Atlantic Forest biome (State of Rio de Janeiro, Brazil), under the authorization of the Ethics Committee on Animal experimentation (protocol 018/2017), and of the Pampa biome (State of Rio Grande do Sul, Brazil), under the authorization of IBAMA/SISBIO (number 33667). From the Atlantic Forest biome, 14 cadavers were collected (seven males and seven females) and from the Pampa biome, 20 cadavers (eight males and twelve females). The collection sites of the specimens of the Atlantic Forest biome were around latitude 22º and those of the Pampa biome, 29º, about 1.520 km in a straight line ( Figure 1). Source: adapted from Google Maps ® After collection, the specimens had their thoracic cavities opened, the thoracic aorta identified for cannula placement, and fixed by intravascular injection of a 50% formaldehyde solution. Next, the latex solution stained with red pigment was perfused in the vascular bed and the cadavers were immersed in polyethylene tanks with a 10% formaldehyde solution to finish the process of fixing and conserving the specimens.
After at least seven days of fixation, the cadavers were washed in running water and the peritoneal cavity was opened for dissection and for measuring the kidneys and renal vessels (Table 1). A digital caliper (0-150 mm, 0.01 mm resolution, accuracy ± 0.02 mm, Eda ® ) was used to obtain measurements of craniocaudal length, lateromedial width, and dorsal-ventral thickness in both kidneys. Renal volume was calculated by applying the equation for the volume of an ellipsoid stipulated by Barr (1990), where Volume (V) = length (L)  Width (W)  Thickness (T) x 0.523. The lengths of the renal artery and vein were also measured in both antimeres, taken between the abdominal aorta or caudal vena cava to the renal hilum. Only kidneys and renal vessels in perfect macroscopic conditions were included in the present study and, therefore, in some cadavers it was not possible to measure both kidneys and vessels bilaterally. To determine skeletopy accurately, the poles (cranial and caudal) of each kidney were marked with radiopaque pins before cadavers were radiographed. The radiographic images were obtained in a lateral projection with the Phillips ® brand device, Aquilla Plus 300 model. Radiographs were taken in a Kodak ® Direct View Computerized cassette system, exposure of 40 KV, 200 mA in 0.1 s and saved in DICOM ® format. After viewing with Radiant Dicom Viewer ® software version 1.6.8, the files were converted to JPEG format. The position of both poles projected to the vertebrae was noted.
The BioEstat 5.3 ® software was used to obtain descriptive statistics data (standard deviation and arithmetic mean) and unpaired Student's t-test to compare measurements between specimens of the two biomes (Atlantic Forest  Pampa), between sexes and antimeres, considered significant when p < 0.05.

DISCUSSION
The ellipsoid volume of the kidney of the C. thous, estimated between 14 cm 3 in the left kidney and 16 cm 3 in the right kidney, was bigger than of L. gymnocercus (12 cm 3 in the left kidney and 11 cm 3 in the right kidney) (SOUZA et al., 2018) and also bigger than in the domestic cat (9-12 cm 3 in the left kidney and 9-11 cm 3 in the right kidney) (STOCCO et al., 2016). Such comparisons seem pertinent to the size of the species: C. thous tends to be smaller than a medium-sized dog, but slightly bigger than L. gymnocercus and a lot bigger than domestic cats and M. p. furo.
Among the data of the present study, probably what is most striking is the fact the kidneys of the specimens of C. thous from Pampa biome are significantly bigger in practically all the dimensions than those of individuals from the Atlantic Forest biome. Previous studies have shown that the skull size of C. thous specimens is inversely proportional to temperature-related variables; it means that in populations to the south of Ecuador, the greater the latitude the greater the size of individuals (MARTINEZ et al., 2013). This finding is consistent with the results of the present study, where specimens of C. thous collected near latitude 29º (Pampa) have kidneys significantly bigger than specimens collected near latitude 22º (Atlantic Forest). The collection areas are about 1.500 km away and differ in vegetation and annual average temperatures. The Pampa biome has more challenging periods of low rainfall than the Atlantic Forest, which is known to be humid (PENEREIRO et al., 2018). Perhaps, this relationship between the selective pressure of the environment on kidney morphology can play a role in understanding the differences found in renal dimensions between the two populations.
Based on the geographic distribution previously proposed (CABRERA, 1931), it is possible the two populations analyzed in the present study constitute distinct subspecies: C. t. entrerrianus at Pampa biome and C. t. azarae at Atlantic Forest biome. In agreement with the comparison of skull dimensions performed in a previous study (MARTINEZ et al., 2013), the kidneys also did not differ between individuals of the same subspecies. It has been recognized three heterogeneous groups of C. thous when it comes to the morphometric patterns of the skull, and the specimens of the present study were also in distinct groups (MACHADO; HINGST-ZAHER, 2009).
When analyzing skulls of C. thous, one study added that the skull of the specimens to the south had a larger fixation area for the temporal muscle, and thinner and sharper extra molar or premolar teeth (BUBADUÉ et al., 2016). These characteristics indicate a greater bite force and greater drilling capacity of the prey, suggesting preference for carnivorous diet in individuals to the south. The authors speculate that the sympatry of C. thous with another canid of similar size, L. gymnocercus, creates a demand for competitive adaptations that would not occur in other regions.
Higher protein intake influences renal hemodynamics and increases the glomerular filtration rate, increasing the volume of the glomeruli and resulting in increased volume and renal mass in several mammal species, including rats, dogs and humans (FINCO, 1999;HAMMOND;JANES, 1998;SCHRIJVERS et al., 2002). Thus, it can be speculated that the kidneys of C. thous populations to the south are bigger not only in proportion to body size but also in adapting to a diet with a higher protein content than individuals in lower latitudes. The confirmation of this hypothesis would depend on a meta-analysis of the diet of these groups and comparative renal histomorphometry or serum biochemistry exams, or both, which are beyond the scope of this study.
The significant differences found in the renal dimensions between individuals of the same species from different biomes generate one more aspect to be considered in the cautious interpretation of the findings of imaging and necropsy examinations in veterinary practice. In addition to geographical origin, age and diet may eventually interfere with renal dimensions, and should be considered while interpreting. The sample group of the present study is composed of young free-living adults, while captive animals can have a differentiated diet and live substantially longer.
Renal dimensions did not differ significantly between antimeres in C. thous, as reported in domestic dogs (SAMPAIO; ARAUJO, 2002) and New Zealand rabbits (SANTOS-SOUSA et al., 2015), and different from human species, whose left kidney is significantly bigger (FERNANDES et al., 2002;MANDARIN-DE-LACERDA, 1989). In L. gymnocercus, the left kidneys had a tendency to be longer and wider than the right ones, although the difference was not significant (p = 0.07 and p = 0.06, respectively) (SOUZA et al., 2018). In domestic cats, only males had the left kidney bigger (STOCCO et al., 2016).
Most studies that present renal dimensions in dogs have methodology based on imaging methods. Some suggest indexes where the kidneys of male dogs are significantly bigger than of the females (LEE; LEOWIJUK, 1982;LOBACZ et al., 2012;MARESCHAL et al., 2007;ARAUJO, 2002;SOHN et al., 2016). In C. thous, no significant difference was found in the renal measures between sexes, when all specimens were compared and when separated by biomes. The absence of differences in renal dimensions between sexes was also documented in L. gymnocercus (SOUZA et al., 2018), New Zealand rabbits (SANTOS-SOUSA et al., 2015) and in human species (CHEONG et al., 2007;EMAMIAN et al., 1993).
In C. thous, the skeletopy of the cranial pole of the right kidney predominated at L1 level, while the left kidney in L2, that is, the right kidney, has a more cranial position. This positioning makes the kidneys of C. thous, especially the left one, free of costal coverage which would facilitate palpation of the abdominal wall during physical examination and its visualization in imaging diagnostic tests. This positioning was identical to what was described in domestic dogs and cats, including the right kidney located at the T13 level in some dogs (EVANS; LAHUNTA, 2013). According to a previous report, the feline renal skeletopy would be more caudal than in dogs and C. thous, with the cranial pole of the right kidney predominating at the level of L2 and the left at the level of L3 (STOCCO et al., 2016). In Mustela putorius furo, the cranial pole of the right kidney is at the level of T14 and the left kidney at the level of L2 (EVANS; AN, 2014). In New Zealand rabbits, the male's cranial poles of right kidneys predominated at level T13, while female's predominated at L1, and left kidneys at level L2 in males and L3 in females (SANTOS-SOUSA et al., 2015). The cranial advancement of the right kidney is common to all domestic mammals, except in pigs (KONIG; MAIERL; LIEBICH, 2016).
In C. thous, LLV was significantly bigger than LRV, which is explained by the greater distance from the left kidney in relation to the caudal vena cava. This finding was identical in domestic dogs (EVANS; LAHUNTA, 2013), L. gymnocercus (SOUZA et al., 2018), domestic cats (STOCCO et al., 2016) and New Zealand rabbits (SANTOS-SOUZA et al., 2015). For the same reason, it would be expected that LRA was bigger than LLA, which did not occur in C. thous, in L. gymnocercus (SOUZA et al., 2018), not even in domestic felines (STOCCO et al., 2016), although mentioned in the domestic dog (EVANS; LAHUNTA, 2013). On the other hand, in New Zealand rabbits, LLA was significantly bigger than LRA (SANTOS-SOUZA et al., 2015). In absolute dimensions, the renal arteries of C. thous were about 5 mm smaller than those of L. gymnocercus (SOUZA et al., 2018), while the renal veins practically did not differ.

CONCLUSIONS
Finally, it can be concluded that the average size of the kidneys of C. thous differed in the two disjoint populations analyzed. This result reinforces the findings of measurements previously performed on the skulls of this species and by other factors to be clarified, such as the differences in protein or hydric intake. Veterinary procedures concerning the kidneys of this species should consider possible differences in size, depending on the habitat, captivity or age of the specimen.