EVALUATION OF THE SERUM BIOCHEMISTRY AND HISTOPATHOLOGY OF KIDNEY AND BLADDER OF DOGS WITH Leishmania sp. IN THEIR URINE AVALIAÇÃO DA BIOQUÍMICA SÉRICA E DA HISTOPATOLOGIA DE RIM E BEXIGA EM CÃES COM Leishmania sp. NA URINA

The visceral establishment of Leishmania infantum in dogs may result in kidney and bladder tissue injury, with L. infantum ending up in urine. This study therefore aimed at investigating the presence of Leishmania sp. in urinary sediments, and correlating the results with those from renal and bladder serum biochemistry and histopathology. Thirty dogs with negative Nested-Polymerase Chain Reaction (PCR) for E. canis were used in the experiment, and were divided into three groups: control group (10 dogs), neither leishmaniasis nor clinical changes; group I (15 dogs), leishmaniasis but no Leishmania sp. in urine; and group II (5 dogs), leishmaniasis, as well as Leishmania sp. in urine. All animals were submitted to clinical, serological, and parasitological diagnosis for leishmaniasis, biochemical exams, and kidney and bladder histopathology. The parasite was also detected in the bladder imprint of one group II dog. Group II dogs presented with very low albumin concentrations, low albumin/globulin ratios, and kidney and bladder lesions. In the kidneys, hydropic degeneration, thickened Bowman's capsule, and thickening of the tubular capsule were detected in all dogs with positive urinary sediment. However, no significant difference in these renal changes was observed between groups. The intensity and distribution of bladder inflammatory infiltrates were significantly (p-value < 0.05, Kruskal-Wallis’ and Dunn’s tests) higher in group II dogs, compared with those of the other groups. The presence of Leishmania sp. in the urine of infected dogs appeared to be related to low serum albumin concentrations and more severe bladder lesions.


INTRODUCTION
Depending on the immune status of each dog, natural infection with Leishmania infantum can result in three levels of clinical responses: asymptomatic, mildly symptomatic, and polisymptomatic (GIUNCHETTI et al., 2006;QUEIROZ et al., 2010;MENDONÇA et al., 2015a). This diversity of clinical symptoms represents a serious challenge during the diagnosis of canine visceral leishmaniasis (CVL), as does the difficulty in obtaining a both highly sensitive and specific diagnostic test, mainly in endemic areas, where serological tests present substandard performance in diagnosing infected and reservoir dogs (CASTRO et al., 2012;SANTOS et al., 2014;MENDONÇA, et al., 2017a;MENDONÇA et al., 2017b). Late diagnosis enables parasite spread and expansion, increasing the possibility for multi-organ lesion development, and altering biochemical parameters.
When assessing the serum biochemistry of CVL dogs, it is common to detect hypoalbuminemia, hypergammaglobulinemia, inversion in the albumin/globulin ratio, increased urea and creatinine concentrations, and increased liver enzyme (alanine aminotransferase). Biochemical quantifications are of great value when analyzing organ (liver and kidney) functionality and possible pathology (LOPES; BIONDO;SANTOS, 2007;MENDONÇA;ALVES, 2015b;COSTA et al., 2015). Biosci Renal alterations are quite common in CVL FERREIRA;CAMARGO et al., 2006;GOMES et al., 2008). Interstitial nephritis and tubular alterations are common in CVL dogs, varying from mild inflammatory infiltrates, to severe lesions and loss of function (GOMES et al., 2008;COSTA et al., 2003).
The presence of Leishmania sp. in urine can be related to the severity of the renal lesions, necessitating kidney impairment evaluation via biochemical measurements. It is also necessary to perform histopathology, given that biochemical parameters only detect kidney injury when it affects over 75% of the function of both kidneys (ALBUQUERQUE et al., 2008;ARESU et al., 2013).
In bladder tissues, previous studies found the occurrence of cystitis in dogs with positive serology for leishmaniasis, and the main observed inflammatory infiltrates were lymphohistoplasmacytic and lymphoplasmacytic, followed by macrophage infiltration. Leishmania sp. antigens have also been immunostained in bladder tissues (SANTOS et al., 2013).
Leishmania sp. has been reported in the urine of dogs and humans (MEBRAHTU et al., 1993;RIERA;VALDARES, 1996;MENDONÇA;ALVES, 2015b). However, consequences of the clinical alterations and lesions induced by the parasite on the urine of VL dogs remain unknown.
This study aimed to evaluate the presence of Leishmania sp. in urinary sediments, and correlating the results with the biochemical parameters and kidney and bladder histopathological injuries in dogs naturally infected with Leishmania sp., in order to identify possible changes favoring parasite occurrence in urinary sediment.

Ethical Consideration
The study was performed in the Animal Sanity Laboratory (LASAN) of the Universidade Federal do Piauí (UFPI), using a protocol approved by the Ethics Committee on Animal Experimentation ECAE / UFPI -No. 022/13.

Sample Size
Thirty domestic dogs of different breeds and ages, with negative Nested-Polymerase Chain Reaction (PCR) for E. canis, were used. They were divided into three groups: the control group, which consisted of 10 dogs without physical abnormalities suggestive of CVL, whose serological and parasitological tests were negative for leishmaniasis; group I, which consisted of 15 animals with Leishmania sp. in the bone marrow, popliteal lymph nodes, and/or skin, but not the urinary sediment; and group II, which consisted of 5 animals with Leishmania sp. in the bone marrow, popliteal lymph nodes, and/or skin, as well as the urinary sediment. The dogs were sacrificed to obtain their kidneys and bladders for histopathological tests. All dogs originated from the Control Center of Zoonoses, without previous clinical evaluation and tests for VL.

Serological Tests and Biochemical Analyses
To perform these tests, 10 mL of venous blood was collected from the jugular, using vacuum collection tubes without anticoagulant, to obtain serum for the TRDPP® CVL serological tests (Bio-Manguinhos kit, Rio de Janeiro, Brazil), the immunosorbent assay (ELISA) -(Bio-Manguinhos kit, Rio de Janeiro, Brazil), and the serum quantifications of urea, creatinine, albumin, and total proteins, performed using the Lab Test Liquiform kit (Lagoa Santa, Minas Gerais, Brazil) and the semi-automatic biochemical analyzer (TP ANALYZER), Thermo Plate. The serological and biochemical tests were carried out following the manufacturer's instructions. The serum globulin concentration was calculated by subtracting albumain from the total protein. The results of the biochemical analyses were compared to the reference values described by Kaneko, Harvey and Bruss (1997).
Parasitological Diagnosis for CVL Culture and smear slide using samples of bone marrow, lymphnode, and damaged skin Bone marrow and lymph node punctures were performed with the aid of a 20 mL syringe attached to a 40 x 12 mm needle, and a 10 mL syringe attached to a 25 x 7 mm needle, respectively. The obtained samples were plated on NNN culture medium enriched with Schneider's. The readings for Leishmania sp. promastigote forms were taken on the 5 th , 8 th , 10 th , and 15 th day after seeding. The same bone marrow and lymph node samples were used in smear slides and stained using the Giemsa method, to investigate the Leishmania sp. amastigote forms. In dogs with skin lesion, a scrap was collected and stained using the Giemsa method.

Culture and smear slide using urinary sediment
To investigate Leishmania sp. in the urine sediments, volumes ranging from 6 to 80 mL of urine were collected by cystocentesis. The samples were then centrifuged at 3,600xg for 20 min, and the obtained sediments seeded in NNN culture medium supplemented with Schneider's, to identify Leishmania sp. promastigote forms. Readings were taken on the 5 th , 8 th , 10 th , and 15 th day after seeding. Two microliters of urine sediment were spread on slides and stained using the Giemsa method, to investigate the Leishmania sp. amastigote forms.

Tissue collection for cytology and histopathology
The dogs were euthanized following the rules of the National Board of Animal Experimentation Control (CONCEA), Law No. 11,794 of 2008, October 8 th and Resolution No. 1000, of May 11, 2012, and according to the following procedure: sedation with acepromazine 0.2% at a dose of 0.5 mg/kg/LW, general anesthesia with Ketamine chloride 10% at a dose of 15 mg/kg/LW, and subsequent application of potassium chloride to 10% at a dose of 100 mg/kg/LW, both administered intravenously.

Cytology
Imprint slides were preapared with kidney and bladder tissues, and then fixed in methyl alcohol. After complete alcohol evaporation, staining was performed with Giemsa at a 1:5 dilution in distilled water, for 40 min. The visualization was performed with a 100x optical microscope, to investigate Leishmania sp. amastigote forms.

Histopathology
About 2 cm-thick kidney and bladder fragments, fixed in 10% buffered formalin were then dehydrated, diaphanized, and embedded in paraffin to form 5μm-thick histological sections, which were partially performed in series. The sections were deparaffinizaed in xylene for 10 min and hydrated in decreasing concentrations of ethanol for 5 min. The kidney sections were stained using hematoxylin and eosin (HE), Periodic Acid Schiff (PAS), and Masson trichrome, while the bladder sections underwent only HE, and were examined under a light microscope.
The intensity of the inflammatory infiltrates was rated on a scale from 0 to 4, according to Tisher and Brenner (1994) description and distribution ranging from 0 to 3: where, 0 = absent, 1 = focal (a focal inflammatory infiltrate), 2 = multifocal (more than one focal inflammatory infiltrate) and 3 = diffuse (inflammatory infiltrate scattered in the whole or largely distributed in the sections). Degenerative changes and the location and cell type of the inflammatory infiltrate were classified only as absent or present.

Nested-PCR for E. Canis
A 200 µL blood sample was subjected to DNA extraction using the commercial kit "QIAamp DNA Mini Kit -Qiagen", following the manufacturer's instructions. The Nested-PCR was performed using the following reagents: 10 x PCR buffer, 50 mM MgCl 2 , 10 mM dNTP Mix, and Platinum TaqDNA Polymerase. In the first reaction, the primers used were EHO F -5´-AGAACGAACGCTGGCGGCAAGCC-3' e EHO R -5´-CGTATTACCGCGGCTGCTGGC-3' (DAWSON et al., 1994), specific to the 16S rRNA segment of the genus Ehrlichia. In the second reaction, the primers ECA F 5´ -CAATTATTTATAGCCTCTGGCTATAGGAA -3' (YABSLEY et al., 2004) and ECA R -5' -CGTATTACCGCGGCTGCTGGC -3' (DAWSON et al., 1994) specific to the 16S rRNA segment of E. canis were used. Amplification was performed in a "Life Pro Thermal Cycler" using the following temperature/time: initial denaturation step at 94°C for 10 min, then 40 cycles of denaturation at 94°C for 60 s, primer annealing at 60°C for 60 s, and then primer extension at 72°C for 60 s. The final step was primer extension at 72°C for 4 min, and then maintainance at 4°C (BULLA et al., 2004). For the negative control, DNA from dogs known to be negative for Ehrlichia sp., were used. For the positive control, DNA samples from dogs known to be positive for E. canis and a DH82 cell culture DNA sample infected with E. canis, were used. The DNA amplicons were visualized after staining with ethidium bromide with the aid of a UV transilluminator (BioAgency). and photographed in photodocumenter (Bio -Imaging Systems).

Statistical Analyses
The number of clinical signs of groups I and II were compared using the Mann-Whitney test. To compare the biochemical parameters and the intensity and distribution of inflammatory infiltrates observed from kidney and bladder hitopathology tests (control, group I, and group II), Kruskal-Wallis H test, followed by Dunn's post test were performed. Degenerative changes observed in the kidney hitopathological test were compared between groups I and II, using Fisher's exact test. All tests admitted error probability of 5% and were performed using the GramPad prism® version 5.0 (CA, USA).

RESULTS
Of the 30 dogs used, 20 were infected, with five having Leishmania sp. in their urine sediment cultures (Fig. 1A). Only one animal was positive in the bladder imprint (Fig. 1B), belonging to the group of dogs with urinary Leishmania sp. The kidney tissue imprints and urine sediment smears were negative for Leishmania sp.. Regarding the biochemical parameters, the values of albumin and the albumin/globulin ratio of animals with urinary Leishmania sp. were significantly lower (p-value < 0.05, Kruskal-Wallis or Dunn's test) compared with those of infected dogs with negative urine sediments, and the control group (Table 1). No significant difference was observed in the urea and creatinine parameters among the three groups of dogs studied (Table 1).
In evaluating the number of clinical signs in groups I and II, it was observed that dogs with the urinary parasite presented with means 7.8 ± 3.3 higher than those of the infected non-urine Leishmania sp. dogs; 5.1 ± 2.6. However, no significant difference was observed (p-value = 0.12, Mann-Whitney test).
Histopathological analyses detected the following alterations in the kidney: hydropic degeneration, formation of hyaline casts, thickened Bowman's capsule, thickening of the tubular capsule, fibrosis, tubular atrophy, and tubular dilatation (Figures 2A, B, C, and D).
All animals examined had at least one kidney alteration, and were in hydropic degeneration, with thickened Bowman's capsule and thickening of the tubular capsule detected in all dogs with positive urinary sediments (Table 3). However, no significant difference was observed in these renal changes, between the groups.
The positive dogs also presented with inflammatory infiltrates in the kidney and bladder urinary sediments (Figures 3A and B). All presented predominance of macrophages and lymphocytes, and one showed a small amount of plasma cells (Table 4)     In both organs (kidney and bladder), the intensity of the infiltrates ranged from minimal to average, and the distribution, focal to multifocal (Table 4). In the dogs which presented with Leishmania sp. in their urinary sediment, renal inflammatory infiltrates were located in all regions (interstitial, perivascular, and periglomerular). In bladder tissue, inflammatory infiltrates were present more often in the mucous layer, and mainly composed of macrophages and lymphocytes, and to a lesser extent, neutrophils and plasma cells ( Table  4). The infiltrates of plasma cells and neutrophils, when present, presented a small number of cells.
No significant difference was observed among the three groups upon evaluating occurrence, intensity, distribution, location, and kidney tissue inflammatory infiltrate cell type. However, the presence of Leishmania sp. in urinary sediments was significantly related to the occurrence, intensity, and distribution of inflammatory infiltrates in bladder tissues (Table 5 and Fig. 4). No significant difference was observed in the location and cell type of the bladder inflammatory infiltrates, between the three groups. There was also no significant difference in degenerative kidney changes between the infected urine-negative animals and infected urine-positive animals (Table 5).

DISCUSSION
Some studies have reported the viability of Leishmania sp. in urinary sediment in both dogs and humans (MENDONÇA; BATISTA; ALVES, 2015b;MEBRAHTU et al., 1993;RIERA;VALADARES, 1996). However, this is the first study to report investigating the possible causes of the presence of Leishmania sp. in the urine of VL dogs.
Of the 30 dogs in which kidney and bladder imprints were performed, only one was positive in the bladder, with none in the kidney. The positive imprint dog also had Leishmania sp. in its urine, Biosci suggesting that Leishmania sp. reaches the urine through the bladder. Additionally, a study by Santos et al. (2013) observed immunostained Leishmania sp. in 32% of the bladder samples, and in only 8% of the kidney samples.
The severe hypoalbuminemia in dogs with urine Leishmania sp. suggests that the renal lesions were sufficiently severe to ochestrate albumin leakage (BASTOS; BREGMAN; KIRSZTAJN, 2010). According to Manna et al. (2008), renal alterations such as glomerulonephritis, interstitial nephritis, and deposition of immunocomplexes, may lead to a decrease in peritubular perfusion, with consequent macromolecule passage, justified as loss of albumin through the urinary system. Increased albumin passage through the kidneys occurs when there are lesions in this organ, with increased permeability (RUSSO;BAKRIS;COMPER, 2002). This may justify the passage of Leishmania sp. into urine, since albumin is considerably large and cannot be filtered through an unaltered kidney (RUSSO;BAKRIS;COMPER, 2002).
However, the presence of the parasite in urine had no relationship with kidney failure, as indicated by an increase in serum urea and creatinine. This was proven by the results of the urinary sediment culture and biochemical tests, since four of the five animals with Leishmania sp. growth in their urinary sediment cultures presented with normal serum urea and creatinine concentrations, and only one presented with a high serum urea concentration; therefore, there was no renal failure in the dogs of this group.
An elevated urea concentration cannot solely characterize renal failure, since urea concentration is affected by extra-renal factors such as high protein intake and prolonged fasting. It should be evaluated alongside the creatinine concentration, as it is more specific than urea in the analysis of renal function, characterized by renal insufficiency; both creatinine and urea are increased in blood circulation (LOPES; BIONDO;SANTOS, 2007).
Although urea and serum creatinine quantification revealed no renal insufficiency in dogs with positive urine tests, all five presented histopathological lesions in their kidneys. Furthermore, serum albumin levels of all five were very low and four of them showed formation of hyaline casts in renal tubules, attributable to lesions in these organs, with increased vascular permeability and consequent loss of protein through kidney tubules.
The occurrence of renal lesions without alterations in serum creatinine and urea concentrations may be due to recent infection, and consequently low intensity of lesions . Hence, the possibility of Leishmania sp. reaching urine through the kidneys cannot be ruled out because dogs with Leishmania sp. in their urine could be carriers of a recent infection with lesions, without decreased glomerular filtration rates. The chronic character of the renal changes in CVL is evidenced by the increased serum, urea, and creatinine. However, the increase of these biochemical parameters occurs only after deposition of immunocomplexes in the kidneys (SOUSA et al., 2011) and considerable loss of approximately 75% of the nephrons of both kidneys (LANIS et al., 2008).
Although there is the possibility of the passage of Leishmania sp. through the kidneys, we found more evidence that the parasite reaches urine through the bladder. In addition to the evidence presented by Santos et al. (2013), who found a much higher percentage of Leishmania sp. immunostained in the bladder compared to the kidney, we detected a positive bladder imprint in a dog having the parasite in its urinary sediment. In addition, we observed that the intensity and distribution of inflammatory infiltrates in the bladder of dogs presenting with Leishmania sp. in urine were significantly more severe in relation to infected dogs having no parasites in their urinary sediment. Hypoalbuminemia might play a role, as it increases vascular permeability.

CONCLUSION
The presence of Leishmania sp. in urine was related to the low concentration of serum albumin and the high degree of bladder lesions. This is the first study to report the occurrence of Leishmania sp. in a bladder imprint. This finding shows that histopathological lesions in the bladder are caused by the presence of the parasite. Further, in more intense bladder injuries, Leishmania sp. reaches the urine. Finally, CVL can be diagnosed using a urine sediment culture.