Demonstration of Mitochondrial Damage and Mitophagy in Cisplatin-Mediated Nephrotoxicity.

Cisplatin is a chemotherapeutic widely used in the treatment of various types of solid tumors. Acute kidney injury is the most critical dose-limiting factor in cancer patients treated with cisplatin; mitochondrial dysfunction and resultant cell damage by reactive oxygen species released from damaged mitochondria are suspected to be involved in the kidney injury. Pathological features of mitochondrial damage in relation to cisplatin-mediated nephrotoxicity, however, is not fully described. The purpose of this study was to demonstrate mitochondrial damage and clearance of damaged mitochondria by mitophagy in cisplatin-mediated nephrotoxicity. Three groups of rats received a single intraperitoneal injection of cisplatin at 20 mg/kg and were sacrificed at 24, 48 and 72 hours after the treatment. A time-dependent increase in the number of damaged renal tubules and the serum levels of blood urea nitrogen, creatinine, and mitochondrial aspartate transaminase was observed in rats after the treatment. We showed the increased numbers of swollen and fragmented mitochondria, observed by electron microscopy, and of cytochrome c oxidase IV- and 8-nitroguanosine-positive intracytoplasmic granules, detected by immunohistochemistry, in the degenerated renal tubules of the treated animals. Moreover, activated autophagy process was indicated in the degenerated renal epithelial cells, based on the findings of immunohistochemistry of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and lysosomal-associated membrane protein 1 (LAMP-1), a lysosome marker, and swollen and fragmented mitochondria in autophagosomes. These results suggest that mitochondrial damage and clearance of damaged mitochondria by mitophagy is involved in cisplatin-mediated nephrotoxicity.

Quantitative analysis of CD3ε in a cloned canine lymphoma cell line by selected reaction monitoring assay.

We established a mass spectrometry-based quantitative method of assaying CD3ε, a component of the T-cell receptor complex. It revealed a CD3ε level of 1 mol per cell in a newly derived canine T-cell lymphoma cell line. Our results suggest that this method has sufficient sensitivity to quantify CD3ε levels in canine lymphoma cells reliably.

Colestimide improves glycemic control via hepatic glucose production in db/db mice.

The objective of this study was to assess the chronic effects of a bile acid sequestrant, colestimide, on glucose metabolism. After db/db mice were fed a diet containing colestimide or cholic acid (CA) for 12 weeks, we investigated the impact of these agents on glucose and lipid metabolism. Colestimide significantly reduced the elevated fasting blood glucose level (p<0.01), and CA even more markedly reduced fasting blood glucose. The blood glucose level after an oral glucose load was significantly lower in the CA group than in the control group, but the colestimide group showed no significant difference. The insulin response to a glucose load was abolished in the control and colestimide groups. A hyperinsulinemic-euglycemic clamp study revealed that colestimide significantly improved the GIR (p=0.013). Hepatic EGP and Rd were also improved by colestimide, suggesting that it alleviated insulin resistance by suppressing hepatic glucose production and increasing peripheral glucose usage. CA significantly increased both the weight and cholesterol content of the liver, while colestimide reduced these parameters. Colestimide suppressed hepatic gene expression of SHP, but enhanced SREBP2 expression. On the other hand, CA increased the expression of SHP and lipogenic enzymes such as ACC and SCD-1, but had no effect on SREBP2. The present study demonstrated that colestimide improves hyperglycemia and hyperlipidemia, as well as reducing the hepatic lipid content. In contrast, CA exacerbates hyperlipidemia and increases the hepatic lipid content, although it improves glycemic control. Thus, colestimide is a well-balanced drug for the treatment of diabetes mellitus.

Antitumor effect of bevacizumab in a xenograft model of canine hemangiopericytoma.

Canine hemangiopericytoma (CHP) is characterized by frequent local recurrence and increased invasiveness. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in tumors. The aim of the present study was to investigate the effect of a single dose of bevacizumab on a xenograft model of CHP. VEGF protein was secreted from cultured CHP cells and interacted with bevacizumab. Bevacizumab treatment suppressed tumor growth by inhibiting tumor angiogenesis, whereas no significant differences were observed in the proliferation index and apoptosis rates of treated and untreated mice. Thus, bevacizumab had antitumor effects in a xenograft model of CHP.

Pathological study of proximal tubule mitochondria in diclofenac-induced acute kidney injury model mice.

Diclofenac, a non-steroidal anti-inflammatory drug, reportedly targets mitochondria and induces nephrotoxicity via reactive oxygen species. However, there are few detailed reports of pathological analyses of mitochondria and the factors that cause acute kidney injury (AKI) as a result of nephrotoxicity. In this study, we investigated mitochondrial damage in the proximal tubule in AKI mice at 6, 12, and 24 h after administration of diclofenac. Statistical analysis of immunohistochemistry results confirmed that expression of p62 and LC3, which is associated with autophagy, reached a maximum level in the degenerated proximal renal tubule 12 h after diclofenac treatment, with high autophagy activity. Electron microscopy images provided clear evidence that confirmed mitochondrial degeneration and injury as well as autophagy (mitophagy) in mitochondria treated with diclofenac. The purpose of this study was to pathologically characterize both mitochondrial damage in the proximal renal tubules induced by diclofenac and the course of mitophagy to remove the damaged mitochondria. This report provides important information regarding mitochondrial damage in the proximal tubules in diclofenac-induced nephropathy.

Ganglioside GM3 deficiency enhances mast cell sensitivity.

Mast cells are a significant source of cytokines and chemokines that play a role in pathological processes. Gangliosides, which are complex lipids with a sugar chain, are present in all eukaryotic cell membranes and comprise lipid rafts. Ganglioside GM3, the first ganglioside in the synthetic pathway, is a common precursor of the specifying derivatives and is well known for its various functions in biosystems. Mast cells contain high levels of gangliosides; however, the involvement of GM3 in mast cell sensitivity is unclear. Therefore, in this study, we elucidated the role of ganglioside GM3 in mast cells and skin inflammation. GM3 synthase (GM3S)-deficient mast cells showed cytosolic granule topological changes and hyperactivation upon IgE-DNP stimulation without affecting proliferation and differentiation. Additionally, inflammatory cytokine levels increased in GM3S-deficient bone marrow-derived mast cells (BMMC). Furthermore, GM3S-KO mice and GM3S-KO BMMC transplantation showed increased skin allergic reactions. Besides mast cell hypersensitivity caused by GM3S deficiency, membrane integrity decreased and GM3 supplementation rescued this loss of membrane integrity. Additionally, GM3S deficiency increased the phosphorylation of p38 mitogen-activated protein kinase. These results suggest that GM3 increases membrane integrity, leading to the suppression of the p38 signalling pathway in BMMC and contributing to skin allergic reaction.

A porcine lymphoma-derived cell line co-expressing IgM, IgG and IgA.

A cell line (PL38PB) was established from blood samples of a 6-month-old pig that was diagnosed with lymphoma with CD5 expression. Histopathological examination revealed neoplastic lesions in the spleen, liver and lymph nodes. Tumor cells were immunohistochemically positive for CD20 and immunoglobulin heavy chains (µ, γ and α). Membranous CD5 and cytoplasmic Immunoglobulin M (IgM), ​Immunoglobulin G (IgG) and ​Immunoglobulin A (IgA) were detected in PL38PB cells by flow cytometry. In addition, the cytoplasm of PL38PB cells were positive for IgM, IgG and IgA by immunofluorescent. However, no Ig secretion was detected in culture supernatant by Ouchterlony gel diffusion method. Results suggest that PL38PB cells express three Ig isotypes that are produced but not secreted.

Expression of L-type amino acid transporter 1 in canine and feline intracranial tumors.

L-type amino acid transporter 1 (LAT1) is upregulated in various malignant tumors in humans. LAT1 expression correlates with the grade of cancer and prognosis. LAT1 is responsible for the supply of many essential amino acids to cancer cells. Inhibition of LAT1 reduces the amino acids that enter the cell and inhibits cancer cell growth. Therefore, novel anticancer drugs targeting LAT1 have attracted much attention in recent years. In this study, to explore the applicability of using LAT1 expression in intracranial tumors as a prognostic factor and therapeutic target, we investigated the expression of LAT1 in surgically resected primary and secondary intracranial tumor tissues from dogs and cats. Immunohistochemical analysis of LAT1 was performed on intracranial tumor tissue from 14 dogs and 3 cats. Primary intracranial tumors were seen in 10 dogs and included meningiomas, histiocytic sarcomas, pituitary tumors, and gliomas, and 9 out of 10 cases were positive for LAT1. Primary intracranial tumors were seen in 2 cats and included meningioma and lymphoma; both cases were positive for LAT1. Secondary intracranial tumors were positive for LAT1 in 3 out of 4 cases in dogs and 1 out of 1 in cats. Since the majority of intracranial tumors in dogs and cats were positive for LAT1, immunostaining for LAT1 is expected to be a prognostic indicator and therapeutic target in the future.

Progression of glomerulonephritis to end-stage kidney disease in a cat with nephrotic syndrome.

A percutaneous renal biopsy was performed on a 3-year-old female Japanese domestic cat with pleural effusion, mild azotemia, hypoalbuminemia, hypercholesterolemia, and proteinuria. Glomerular lesions included mild diffuse hypercellularity and numerous capsular adhesions with segmental sclerosis/hyalinosis of glomerular tufts. Electron microscopy revealed many subendothelial dense deposits with characteristic outer protrusion of glomerular basement membrane. Diffuse and global granular deposits of IgG and C3 were detected along the capillary walls. Tubulo-interstitial changes were mild at the time of biopsy, but progression of the disease was predicted because of the many capsular adhesions of the glomerular tufts. The cat was fed a prescription diet without any other specific or symptomatic therapy after renal biopsy, and died 43 weeks after the biopsy. At necropsy, extensive tubulo-interstitial fibrosis and mononuclear cell infiltration had developed throughout the cortex and outer medulla, and most glomeruli had extensive global sclerosis or obsolescence with less prominent depositions of IgG and C3.

Equine sarcoid of the glans penis with bovine papillomavirus type 1 in a miniature horse (Falabella).

A 23-year-old Falabella gelding kept in Tochigi, Japan, for more than 20 years presented with a recurrent mass of the glans penis that was first noticed about a year earlier. Partial phallectomy was performed with no adjunctive therapy for local regrowth of the mass. The horse was euthanized 3 months after surgery for urinary retention due to suspected regrowth. The resected mass affected the genital and urethral mucosa of the glans penis, and was diagnosed as equine sarcoid by histopathology and identification of bovine papillomavirus (BPV) DNA. Phylogenetic analysis of the BPV genome of the sarcoid showed high sequence homology to BPV type 1 (BPV-1) from Hokkaido, Japan, suggesting a geographical relationship for BPV-1 in Japan.

Identification of Legionella londiniensis isolated from hot spring water samples in Shizuoka, Japan, and cytotoxicity of isolates.

As part of an epidemiological study on legionellosis, we attempted to isolate Legionella spp. from hot spring water and were able to isolate L. londiniensis HYKF-90505 (=JCM 16338), confirming that L. londiniensis inhabits hot spring water in Japan. To investigate the disease potential of L. londiniensis, we examined its ability to grow intracellularly within Acanthamoeba sp. JAC/E1 strain. The isolated HYKF-90505 was able to grow within Acanthamoeba sp. JAC/E1 strain, and we confirmed also that the HYKF-90505 strain showed cytotoxicity for cultured cells such as J774.1 (JCRB0018). However, in a culture of human U937 cells, the bacterial count was not increased by the intracellular growth of the HYKF-90505 strain. Cells infected for 24 h and stained using the Giménez method showed no intracellular growth of the HYKF-90505 strain. Thus, the isolate appears to be weakly pathogenic to humans.

Early-onset podocyte injury and glomerular sclerosis in osborne-mendel rats.

Progressive glomerular injury associated with early-onset proteinuria was investigated in male Osborne-Mendel (OM) rats aged 5 to 20 weeks. Age-matched male Fischer 344 (F344) rats were used for comparison. OM rats developed mild hypertension and selective proteinuria (albuminuria) from 5 weeks of age, and non-selective proteinuria from 7 weeks of age. Light microscopy of OM kidney revealed hyaline droplets in the podocyte at 5 weeks of age and vacuolation of podocytes and adhesion of the capillary loop to the Bowman's capsule at 7 weeks of age. Segmental glomerulosclerosis developed in OM rats from 15 weeks of age, and global sclerosis appeared at 20 weeks of age. Desmin, a marker of podocye injury, was expressed in podocytes from 10 weeks of age, and the intensity of expression increased with age. Ultrastructurally, damage to podocytes such as effacement of foot processes, decreasing number of filtration slits, and rearrangement of the actin cytoskeleton were observed from 5 weeks of age in OM rat. Glomerular volume in OM rats increased with age and was consistently higher than in age-matched F344 rats. The number of WT-1-positive podocytes and vimentin-positive podocyte area were lower in OM rats and decreased with age. These findings suggest that glomerulonephropathy in male OM rats is associated with glomerular hypertrophy, progressive podocytopathy, and a reduction in podocyte number and area. Renal injury in OM rats was associated with development of early-onset proteinuria and was more progressive than in age-matched F344 rats.

Pancreatic adenosquamous carcinoma with invasion to the spleen in a cat.

A four-and-a-half-year-old female Scottish Fold cat underwent partial pancreatectomy with en-bloc splenectomy. The resected specimen was a biphasic tumor that was diagnosed histologically and immunohistochemically as pancreatic adenosquamous carcinoma (ASC), a ductal carcinoma variant according to the WHO classification of tumors in humans. There was a gradual transition between the adenocarcinoma component and the squamous cell carcinoma component. The squamous cell carcinoma component comprised approximately 30-40% of the tumor. A pancreatic tumor infiltrated into the gastrosplenic ligament and spleen with regional lymph node and mesenteric metastases. Pancreatic ASC has not been reported in animals. This is a case report of feline pancreatic ASC with splenic involvement.

Plasma lipoprotein profile from nonalcoholic steatohepatitis model rats.

We recently revealed that increases in particle sizes of very-low-density lipoproteins (VLDL) are highly correlated with the progression of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), and VLDL particle size may be a minimally invasive indicator of these hepatic disorders. Methionine and choline-deficient (MCD) diet fed animals are usually used as a NASH model; however, the application of this minimally invasive biomarker in MCD diet fed animals remains unclear. In the present study, we measured the levels of liver disease markers and plasma lipoprotein profiles in MCD diet fed rats, and compared them with those of normal diet fed rats. Assessing lipoprotein profiles showed marked increases in VLDL particle sizes in MCD diet fed rats with pathologically and biochemically NASH-like features.

Pathogenesis of liver lesions in Theileria orientalis-inoculated cattle and severe combined immunodeficiency mice with bovine erythrocyte transfusion.

Theileria orientalis (T. orientalis) is a bovine protozoal disease similar to malaria in humans. Although the common outcome of malaria in humans and T. orientalis infection in cattle is hepatic disorder, the mechanisms of its development remain unknown. In this study, we investigated hepatocyte injury characterized by accumulation of macrophages with ingested erythrocytes in sinusoid and extramedullary hematopoiesis in cattle and mice experimentally infected with T. orientalis (T. orientalis-infected cattle and T. orientalis-infected mice). Vacuolization of hepatic cells was frequently observed in the vicinity of the aggregated macrophages in the liver sinusoids of T. orientalis-infected mice. A significant percentage of the macrophages accumulated in the liver sinusoids of the severely infected cattle and mice (14.6% and 24.2 to 53.2%, respectively) reacted positively with interleukin-1, interleukin-6 and TNF-α antibodies. Increase in the production of these cytokines was confirmed in T. orientalis-infected cattle and mice by real-time RT-PCR. These findings strongly suggest that increased cytokine production by the macrophages that have phagocytosed T. orientalis-infected erythrocytes causes hepatic disorder in T. orientalis-infected animals.

A case of canine vasotropic and vasoinvasive nonepitheliotropic lymphoma with unusual tumor cells and extensive dermal necrosis.

A 14-year-old, spayed female Shih-tzu dog presented with masses in the dorsal aspect of cervical region and digit of the right anterior limb. Extensive necrosis was seen in the dermal tissue overlying the tumor, and diffuse round cell proliferation and infiltration were seen histologically from the superficial dermis to the deep dermis. Two types of proliferating cells were present: lymphoblast-like cells with round-oval, vesicular nuclei and moderate-large nucleoli; and smaller cells with characteristic irregularly shaped nuclei. Electron microscopy of these smaller cells showed cerebriform, pleomorphic nuclei with a chromatin pattern characteristic of lymphoid cells, as seen in lymphoblast-like tumor cells. Immunohistochemically, both types of tumor cells were positive for CD3. Most vessel walls had been invaded by tumor cells, resulting in extensive dermal necrosis and hemorrhage. Based on these histopathological findings, the tumor was diagnosed as vasotropic and vasoinvasive nonepitheliotropic lymphoma, characterized by a notable presence of unusual tumor cells with irregularly shaped nuclei and extensive dermal necrosis.

Cutaneous clear cell adnexal carcinoma in a dog: special reference to cytokeratin expression.

A 5-year-old, male Bichon-Frise dog presented with a cutaneous mass in the basal region of the auricle. Histologically, the cutaneous neoplasm was comprised of lobules with solid cellular proliferation separated by thin fibrous septa. Neoplastic cells varied in size, with moderate to abundant amounts of PAS-positive cytoplasm, large nuclei and prominent nucleoli. Immunohistochemical examinations showed that tumor cells were positive for pan-cytokeratin (CK) (AE1/AE3 and CAM5.2), CK8 and CK18, but negative for pan-CK (KL1), CK7, CK14, CK16 and CK20. Double-labeled immunofluorescence testing indicated that neoplastic cells frequently co-expressed CK and vimentin, suggesting divergent differentiation of tumor cells. Based on these findings, the tumor was diagnosed as canine clear cell adnexal carcinoma.

Pathological characterization of collagenofibrotic glomerulonephropathy in a young dog.

This report describes the morphological and immunohistochemical findings of a case of apparent collagenofibrotic glomerulonephropathy in a 7-month-old dog. Clinical examination showed moderate protenuria with elevated blood urea nitrogen and creatinine. Histopathological examination of the glomerular capillary walls and mesangial areas revealed diffuse and global accumulation of eosinophilic homogeneous or fine fibrous materials, which were immunohistochemically positive for type III collagen. On electron microscopy, the randomly crossed fibrils had transverse bands with a periodicity of approximately 60 nm. The clinical, histopathological, immunohistochemical and electron microscopic findings of the present dog were consistent with those of the human, childhood form of collagenofibrotic glomerulonephropathy.

Growth in Acanthamoeba sp. and antibiotic susceptibility of Legionella micdadei isolated from hot spring water samples.

As part of an epidemiological study on legionellosis, we attempted to isolate Legionella spp. from hot spring water samples, and were able to isolate Legionella micdadei from 3 (5.5%) of 55 samples. All of these isolates were able to grow within Acanthamoeba sp., suggesting that the isolates will be pathogens. We also confirmed that the K-2 strain from hot spring water grew in guinea pig monocytes. Sensitivity tests using 10 drugs showed that the isolates were most sensitive to imipenem, with the MIC90 of 0.032 microg/ml, were least sensitive to minocycline, with the MIC90 of 4 microg/ml, and were not sensitive to low amounts of other drugs.

Pathological Study on Epithelial-Mesenchymal Transition in Silicotic Lung Lesions in Rat.

Silicosis, caused by the inhalation of crystalline silicon dioxide or silica, is one of the most severe occupational diseases. Persistent inflammation and progressive massive pulmonary fibrosis are the most common histological changes caused by silicosis. Association of epithelial-mesenchymal transition (EMT) of hyperplastic type II epithelial cells with the fibrotic events of pulmonary fibrosis has been suggested in in vitro silica-exposed cultured cell models, patients with idiopathic pulmonary fibrosis, and bleomycin-induced experimental models. Histological features of EMT, however, are not fully described in silicotic lungs in in vivo. The purpose of this study was to demonstrate EMT of hyperplastic type II epithelial cells in the developmental process of progressive massive pulmonary fibrosis in the lungs of rats exposed to silica. F344 female rats were intratracheally instilled with 20 mg of crystalline silica (Min-U-Sil-5), followed by sacrifice at 1, 3, 6, and 12 months after instillation. Fibrosis, characterized by the formation of silicotic nodules, progressive massive fibrosis, and diffuse interstitial fibrosis, was observed in the lungs of the treated rats; the effects of fibrosis intensified in a time-dependent manner. Hyperplasia of the type II epithelial cells, observed in the massive fibrotic lesions, dominated in the lungs of rats at 6 and 12 months after the treatment. Immunohistochemistry of the serial sections of the lung tissues demonstrated positive labeling for cytokeratin, vimentin, and α-smooth muscle actin in spindle cells close to the foci of hyperplasia of type II epithelial cells. Spindle cells, which exhibited features of both epithelial cells and fibroblasts, were also demonstrated with bundles of collagen fibers in the fibrotic lesions, using electron microscopy. Increased expression of TGF-ß was shown by Western blotting and immunohistochemistry in the lungs of the treated rats. These findings suggested that enhanced TGF-ß expression and EMT of hyperplastic type II epithelial cells are involved in the development process of progressive massive pulmonary fibrosis during silicosis.