Sawane Mitani

Comparative study of chronic kidney disease in dogs and cats: Induction of myofibroblasts

We investigated the kidneys of dogs and cats to clarify whether renal myofibroblasts induction is associated with the severity of chronic kidney disease (CKD). Immunohistochemical expression of myofibroblast markers, alpha-smooth muscle actin (SMA) and vimentin, were evaluated quantitatively. The degrees of glomerulosclerosis, glomerular hypertrophy, interstitial cell infiltration, and interstitial fibrosis were also evaluated quantitatively. The plasma creatinine (pCre) concentrations correlated with glomerulosclerosis, cell infiltration, and fibrosis in dogs, and only with fibrosis in cats. The alpha-SMA expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, and with pCre and fibrosis in cats. Tubular vimentin expression correlated with fibrosis in cats, but not in dogs. Interstitial vimentin expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, but only with pCre in cats. In conclusion, it was suggested that the severity of CKD in dogs and cats was mediated by different pathways associated with myofibroblasts expression.

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 frameshift mutation in the canine HEXB gene in toy poodles with GM2 gangliosidosis variant 0 (Sandhoff disease)

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations in the HEXB gene. Toy poodles recently were reported as the second breed of dog with SD. The present paper describes the molecular defect of this canine SD as the first identification of a pathogenic mutation in the canine HEXB gene. Genomic and complementary DNA sequences covering exonic regions of the canine HEXB gene, except exon 1, were analysed using DNA and RNA in an affected dog. A homozygous single base pair deletion of guanine in exon 3 was identified at nucleotide position 283 of the putative open reading frame (c.283delG). This mutation has the potential to cause a frameshift resulting in the alteration of valine at amino acid position 59 to a stop codon (p.V59fsX). Genotyping using the mutagenically separated PCR method demonstrated a correlation between phenotype and genotype in dogs with a pedigree related to the disease and that the mutation was rare in a randomly-selected population of toy poodles. These results strongly suggest that the deletion is pathogenic.

Mutational analysis of the feline CLN3 gene and an ultrastructural evaluation of lysosomal storage materials in a cat with neuronal ceroid lipofuscinosis: An investigation into the molecular basis of the disease

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease caused by a number of different genes. A mutational analysis of the feline CLN3 gene was performed in a cat with NCL that had vacuolated lymphocytes, which is a feature of human NCL caused by defects of the CLN3 gene. To determine the candidate gene(s) responsible for this case, NCL-specific ultrastructures of storage materials were analysed. A sequence analysis indicated that the CLN3 gene was not likely to be responsible for this case of feline NCL because no deleterious mutation was detected. An ultrastructural analysis did not reveal any candidate gene because of inconsistency with any pattern found in human NCL. These findings suggest that the diagnostic criteria for human NCL are not directly applicable to feline NCL.

Dihydropyrimidinase Deficiency: The First Feline Case of Dihydropyrimidinuria with Clinical and Molecular Findings

Dihydropyrimidinase (DHP, EC 3.5.2.2) is the second enzyme of the pyrimidine degradation pathway and a deficiency of this enzyme is responsible for a rare inborn metabolic syndrome characterized by dihydropyrimidinuria. Here we report a cat with DHP deficiency, manifesting malnutrition, depression, vomiting, and hyperammonemia. A gas chromatographic-mass spectrometric analysis of urinary metabolic substances showed the presence of large amounts of dihydrouracil and dihydrothymine and moderate amounts of uracil and thymine, suggesting DHP deficiency. Analysis of the feline DPYS gene encoding DHP demonstrated that the cat was homozygous for the missense mutation c.1303G>A (p.G435R) in exon 8, which corresponds to a known mutation in a human patient with DHP deficiency. Population screening in 1,000 cats did not reveal any animal possessing this mutation, suggesting the prevalence of the mutant allele to be very low. This is the first report of naturally occurring DHP deficiency in animals and the cat represents a model of the human disease.

Rapid and simple polymerase chain reaction-based diagnostic assays for GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japanese domestic cats.

Polymerase chain reaction (PCR)-based assays combined with microchip electrophoresis were developed and evaluated for diagnosis and genotyping of GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japanese domestic cats. A preliminary genotyping survey was carried out in the population of Japanese domestic cats (1,015 cats in total) in southern Japan. Three kinds of assays including PCR primer-induced restriction analysis (PIRA) and mutagenically separated (MS)-PCR were carried out using blood-stained Flinders Technology Associates filter papers (FTA cards) as templates. The PCR products were analyzed by both agarose gel and microchip electrophoreses. All assays were sufficient to determine the genotypes of this disease, but MS-PCR offered the most rapid and simplest test, as it does not need the restriction enzyme step required in PCR-PIRA. The use of microchip electrophoresis in combination with FTA cards for sampling could shorten the time required for genotyping and simplify the procedure as well. The genotyping survey in the current study did not find any cats that possessed the mutant allele, suggesting that the prevalence of this allele is low (<0.1%) in southern Japan.

Age-related histological changes in kidneys of Brown Norway rat.

ABSTRACT In this study, age-dependent histological changes in the kidneys of Brown Norway rat, a strain useful for conducting aging research, were evaluated. Examination was performed at 3, 12, 18, 24 and 30 months of age. Sclerotic and hypertrophic changes of the glomeruli were observed, and quantitative scores of these changes persistently increased with age. A marginal increase in scores was observed for glomerular cystic changes and tubulointerstitial damage. Further, urothelial hyperplasia was observed in the renal papillae, particularly at 30 months of age. In conclusion, the findings of the present study demonstrate that the Brown Norway strain exhibits persistent, but mild progression of age-dependent renal histological changes.

Real-Time PCR Genotyping Assay for GM2 Gangliosidosis Variant 0 in Toy Poodles and the Mutant Allele Frequency in Japan

ABSTRACT GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations of the HEXB gene. In canine SD, a pathogenic mutation (c.283delG) of the canine HEXB gene has been identified in toy poodles. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid and large-scale genotyping and screening for this mutation. Furthermore, a genotyping survey was carried out in a population of toy poodles in Japan to determine the current mutant allele frequency. The real-time PCR assay clearly showed all genotypes of canine SD. The assay was suitable for large-scale survey as well as diagnosis, because of its high throughput and rapidity. The genotyping survey demonstrated a carrier frequency of 0.2%, suggesting that the current mutant allele frequency is low in Japan. However, there may be population stratification in different places, because of the founder effect by some carriers. Therefore, this new assay will be useful for the prevention and control of SD in toy poodles.

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.

Nephron segment identification in the normal canine kidney by using lectin histochemistry

Lectin-binding patterns in normal canine kidneys were histochemically investigated using eight lectins. WGA, ConA, and RCA-I showed positive signals in glomerular capillary walls, with signals for RCA-I being detected heterogeneously. In tubular segments, signals for WGA, s-WGA, ConA, and RCA-I were distributed widely from proximal tubules to collecting ducts, whereas those for SBA, PNA, DBA, and UEA-I were localized in thin limbs of the loop of Henle, thick ascending limbs, distal tubules, or collecting ducts. Apart from PNA and UEA-I, lectins showed heterogeneous bindings in collecting ducts with the heterogeneity. UEA-I-positive reactions were restricted to those parts of the distal tubules in close proximity to the glomeruli, and in these parts, signals in the macula densa were markedly stronger than in other regions. Based on the present findings, lectin probes, singly or in combination, could be utilized to identify the affected nephron segment in canine renal pathology.