Mariko Sawa

A comparative study of chronic kidney disease in dogs and cats: induction of cyclooxygenases.

The present study investigated whether renal cyclooxygenase (COX) induction is associated with the severity of chronic kidney disease (CKD) in dogs and cats. The collected kidneys were examined histopathologically and immunohistochemically. The immunoreactivities of COX-1 and COX-2 were evaluated quantitatively, and the correlations to the plasma creatinine concentrations, glomerular size, glomerulosclerosis, interstitial fibrosis, and interstitial cell infiltration were evaluated statistically. Immunoreactivities for COX-1 were heterogeneously observed in the medullary distal tubules and collecting ducts; no correlations with the severity of renal damage were detected. Immunoreactivities for COX-2 were heterogeneously observed in the macula densa (MD) regions. In dogs, the percentage of COX-2-positive MD was significantly correlated with the glomerular size. In cats, glomeruli with COX-2-positive MD had significantly higher sclerosis scores than those with COX-2-negative MD. In conclusion, renal COX-2 is induced in canine and feline CKD, especially in relation to the glomerular changes.

A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100 protein in veterinary diagnostic cytology.

The objective of this study was to establish a simple and rapid immunocytochemical technique that can be used in veterinary diagnostic cytology. Air-dried impression smears were collected from canine tumors. Samples of epithelial tumors, mesenchymal tumors, and malignant peripheral nerve sheath tumors and melanomas were used for detection of cytokeratin, vimentin, and S-100 protein, respectively. The labeled streptavidin-biotin system was used in the present study. Optimal fixation was determined using standard immunocytochemical procedures, and acetone fixation was found to be the most effective. Optimal concentrations of primary and secondary antibodies were determined at a preset 5-min incubation. Omission of H2O2 treatment, shortening the time for blocking and labeled-streptavidin incubation, and simplifying washing did not decrease immunopositive intensities or enhance false-positive reactions. The described rapid protocol requires approximately 45 min without the use of any special equipment.

Development and application of multiple immunofluorescence staining for diagnostic cytology of canine and feline lymphoma.

BACKGROUND Immunophenotyping of canine and feline lymphoma to determine B-cell or T-cell origin is important for predicting prognosis and for development of treatment protocols. For advanced diagnostic cytology tests that can be performed on smears are required to predict the immunophenotype of lymphomas. OBJECTIVES The aim of this study was to develop a multiple immunofluorescence (MIF) staining method for the determination of lymphocyte immunophenotype in cytologic specimens, and to evaluate its clinical utility. METHODS B cells and T cells were detected using anti-CD79α and anti-CD3 antibodies, respectively, followed by specific fluorescence-labeled secondary antibodies. The MIF staining method was first developed using fresh-frozen sections of normal canine lymph nodes. The optimal fixative, the necessity of antigen retrieval (AR), and the optimal concentration of the antibodies were determined. The MIF method was then applied to smears of normal lymph nodes, and to clinical samples from dogs and cats with lymphoma. The MIF results were compared to genetic clonality results. RESULTS B and T cells were detected based on specific fluorescence in frozen sections, using formalin fixation without AR. Specific fluorescence was also detected in smears from normal lymph nodes and lymphomas, and the immunophenotypes predicted from this MIF staining method completely corresponded to those from genetic clonality analysis. CONCLUSIONS The MIF staining method that we developed in this study effectively distinguished lymphocyte immunophenotypes with high specificity and sensitivity using a single smear sample, and was useful as a diagnostic tool for canine and feline lymphoma.

Intrarenal Distributions and Changes of Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Feline and Canine Chronic Kidney Disease

ABSTRACT Angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin system (RAS). ACE2 is a newly identified member of the RAS. The present immunohistochemical study focused on changes in intrarenal ACE and ACE2 immunoreactivity in feline and canine chronic kidney disease (CKD). ACE immunoreactivity was predominantly observed in the brush border of the proximal tubules in dogs and cats. ACE immunoreactivity was lower in CKD kidneys than in normal kidneys, and quantitative analysis demonstrated negative correlations between ACE and renal tissue damage in dogs. ACE2 immunoreactivity was also detected in the proximal tubules; it increased or decreased with CKD in dogs, depending on the renal region assessed. The changes in ACE and ACE2 in CKD were associated with the plasma creatinine concentration in dogs. Findings from dogs with glomerulonephritis were similar to those from dogs with non-glomerulonephritis. The present study suggests that changes in the intrarenal expression of ACE and ACE2 contribute to the pathological mechanisms of canine CKD, but not to the mechanisms of feline CKD.

Molecular prevalence of multiple genetic disorders in Border collies in Japan and recommendations for genetic counselling

Reproductive management is necessary to prevent deleterious genetic disorders in purebred dogs, but comprehensive studies aimed at prevention of multiple underlying genetic disorders in a single breed have not been performed. The aims of this study were to examine mutant allele frequencies associated with multiple genetic disorders, using Border collies as a representative breed, and to make recommendations for prevention of the disorders. Genotyping of known mutations associated with seven recessive genetic disorders was performed using PCR assays. More than half (56%) of the Border collies had no mutant alleles associated with any of the seven disorders, suggesting that these disorders can be removed from the population over several generations. Since frequencies of each mutant allele differed among disorders, reproductive management should be performed after the establishment of prevention schemes that are appropriate for each disorder, the type and specificity of genetic test available, and the effective population size in each breeding colony.

A Practical Technique for Electron Microscopy of Buffy Coats in Dogs and Cats.

Plastic hematocrit tubes (PHTs) are convenient tools for electron microscopy (EM) of peripheral blood buffy coats, and the PHT‐EM technique is expected to be a practical method for veterinary clinical medicine. In this study, fixatives composed of various concentrations of sucrose, glutaraldehyde, and phosphate buffer (PB) were tested for preparing canine and feline buffy coats. The highest quality images were obtained using a fixative consisting of 2.5% glutaraldehyde in 0.1 m PB, and it was concluded that this method allows clinicians who are inexperienced in histological techniques can conveniently transport buffy coat samples to diagnostic laboratories for analysis by EM.

Rapid multiple immunofluorescent staining for the simultaneous detection of cytokeratin and vimentin in the cytology of canine tumors

BACKGROUND Immunocytochemistry (ICC) is utilized as an advanced technique in veterinary cytology. In tumor diagnosis, cytokeratin and vimentin are markers used to distinguish the origin of tumor cells. Standard enzyme-based ICC has limitations in clinical use; and therefore, more convenient and reliable methods are needed. OBJECTIVES The purpose of this study was to develop a rapid multiple immunofluorescent (RMIF) detection method for dual cytokeratin and vimentin staining on cytology slides in dogs. METHODS Air-dried smear samples from solid tumors and sediments of pleural effusions were prepared from dogs (n = 14) that were admitted to the Veterinary Teaching Hospital, Kagoshima University, Japan. Mouse monoclonal anti-human cytokeratin (AE1/AE3) and rabbit monoclonal anti-human vimentin (SP20) antibodies were used as primary antibodies, followed by staining with Alexa Fluor-conjugated secondary antibodies. Staining using the RMIF method was compared with enzyme-based ICC staining. RESULTS Rapid multiple immunofluorescent immunostaining was clear and specific in the evaluated smears, whereas the enzyme-based ICC showed nonspecific signals. By using the RMIF staining method, epithelial cells, mesenchymal cells, and mesothelial cells could be classified on a single smear of a pleural effusion. In smears of lymph nodes with epithelial tumor metastases, the RMIF method successfully detected metastatic epithelial tumor cells. CONCLUSIONS The RMIF method might be a useful tool for diagnostic cytology in veterinary medicine.

Acquired Fanconi syndrome in two dogs following long-term consumption of pet jerky treats in Japan: case report

Renal Fanconi syndrome has recently been associated with the ingestion of pet jerky treats from China in mostly small breed dogs in North America, Australia and Europe. We report here about two dogs with Fanconi syndrome following pet jerky treats exposure in Japan. A mixed-breed dog and a French bulldog showed weight loss, polyuria and polydipsia. For years, the owners had been feeding large quantities of pet jerky treats containing chicken prepared in China. Diagnostics revealed glycosuria without hyperglycemia, severe aminoaciduria, and in one case also ketonuria, hypokalemia and metabolic acidosis. A diagnosis of Fanconi syndrome associated with long-term consumption of Chinese pet jerky treats was made. Both dogs recovered fully following withdrawal of the pet jerky treats and supportive care. Fanconi syndrome of dogs in association with the consumption of pet jerky treats of Chinese origin can cause a broad proximal tubular defect with glycosuria and generalized amino aciduria, and should be also considered in Asia. Jerky treats associated Fanconi syndrome can be completely reversible following withdrawal of the treats and supportive care to correct the metabolic abnormalities.

Degenerative myelopathy in the Collie breed: a retrospective immunohistochemical analysis of superoxide dismutase 1 in an affected Rough Collie, and a molecular epidemiological survey of the SOD1: c.118G>A mutation in Japan

Canine degenerative myelopathy (DM) is an adult-onset, progressive neurodegenerative disease that occurs in multiple dog breeds. A DM-associated mutation of the canine superoxide dismutase 1 (SOD1) gene, designated as c.118G>A (p.E40K), has been implicated as one of pathogenetic determinants of the disease in many breeds, but it remains to be determined whether the c.118G>A mutation is responsible for development or progression of DM in Collies. Previously, a Rough Collie was diagnosed clinically and histopathologically as having DM in Japan, suggesting the possibility that the Collie breed may be predisposed to DM due to the high frequency of c.118G>A in Japan. In this study, accumulation and aggregate formation of SOD1 protein were retrospectively demonstrated in the spinal cord of the DM-affected dog by immunohistochemical analysis. Furthermore, a molecular epidemiological survey revealed a high carrier rate (27.6%) and mutant allele frequency (0.138) of c.118G>A in a population of Collies in Japan, suggesting that the Collie breed may be predisposed to DM associated with c.118G>A, and the prevention of DM in Collies in Japan should be addressed through epidemiological and genetic testing strategies.

Low expression of cyclooxygenase-2 in chronic kidney disease in young dogs

Chronic kidney disease (CKD) often results in end-stage renal failure in young dogs; however, the pathogenesis of this disease is not established. This study investigated renal expression of cyclooxygenase (COX)-1 and COX-2 proteins in three dogs with chronic kidney disease by immunohistochemistry. Histopathology showed asynchronous differentiation of renal tissues, including immature glomeruli. COX-1 signals were not detected in diseased or normal kidneys. COX-2 signals were low or undetectable in diseased kidneys, while normal kidneys showed clear positive signals in the macula densa (MD). Quantitative scores of COX-2 in diseased kidneys were significantly lower than those in normal kidneys. These findings demonstrate low renal COX-2 expression in CKD in young dogs, but whether this is correlated with disease pathogenesis remains unclear.