Mami Kouchi

A Novel Renal Carcinoma Predisposing Gene of the Nihon Rat Maps on Chromosome 10

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Spra‐gue‐Dawley (SD) strain in Japan, and named the “Nihon” rat in 2000. This study was designed to map the RC susceptibility gene in the Nihon rat using 113 backcross annuals. Our present data clearly show that the Nihon gene is genetically linked to interleukin‐3 (IL3) gene (χ2=93.6, Lod score=25.16), lethal (2) giant larvae (LLGL1) locus (χ2=109.0, Lod score=31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE) gene (χ2=90.6, Lod score=23.87), which are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2) gene (located on the proximal part of rat chromosome 10; human chromosome 16p 13.3)–21.3 cM–IL3 gene (human 5q23‐31)–4.4 cM–Nihon gene–0.9 cM–LLGL1 locus (human 17p11.2)‐4.4 cM–MYHSE gene (human 17pl3.1). We also detected loss of the wild‐type allele at the MYHSE locus, fitting Knudsons “two hit” model. Thus, the Nihon rat should have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.

Natural history of the Nihon rat model of BHD.

Hereditary cancer was first described in the rat by Eker and Mossige in 1954 in Oslo. The Eker rat model of hereditary renal carcinoma (RC) was the first example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal, and has been contributing to the elucidation of renal carcinogenesis. Recently, we found a second hereditary RC model in the Sprague-Dawley (SD) rat, in Japan in 2000, which was named the Nihon rat. The Nihon rat is also an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker rat, which are predominantly of the clear cell type (this type represents approximately 75 % of human RCC), and develop from earlier preneoplastic lesions than the Eker rat. We performed a genetic linkage analysis of the Nihon rat using 113 backcross animals, and found that the Nihon mutation was tightly linked to genes, which are located on the distal part of rat chromosome 10. Finally, we identified a germline mutation in the Birt-Hogg-Dubé gene (Bhd) (rat chromosome 10, human chromosome 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat gene sequence, resulting in a frame shift and producing a stop codon 26 amino acids downstream. Thus, the Nihon rat will contribute to understanding the BHD gene function and renal carcinogenesis.

Serine 62 is a phosphorylation site in folliculin, the Birt-Hogg-Dubé gene product

MINT‐7298229: FNIPL (uniprotkb:Q9P278) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)

Regulation of folliculin (the BHD gene product) phosphorylation by Tsc2-mTOR pathway.

The Birt-Hogg-Dubé gene (BHD) encodes the tumor suppressor protein folliculin (FLCN). The function of FLCN has recently been implicated in the regulation of rapamycin-sensitive mTOR complex (mTORC1). Reciprocally, the mTORC1-dependent phosphorylation of FLCN was reported. However, precise mechanism of FLCN phosphorylation and functional interaction of FLCN with tuberin, the product of tuberous sclerosis 2 gene (TSC2) which is a negative regulator of mTORC1, are unclear. Here we report that multiple phosphorylation in FLCN are evoked by downregulation of tuberin as well as by Rheb expression. We found that phosphorylation at Ser62 and Ser302 are differently regulated by mTORC1-dependent pathway. Some unknown kinases downstream of tuberin-mTORC1 are thought to directly phosphorylate FLCN. Interestingly, our results also suggest that the complex formation of FLCN with AMPK is modulated by FLCN phosphorylation. These results suggest that FLCN is involved in a novel mechanism of signal transduction downstream of tuberin.

The First Case of Feline Infectious Peritonitis-like Pyogranuloma in a Ferret Infected by Coronavirus in Japan.

A male ferret, which was purchased from abroad at 9 months of age, had shown significant weight loss starting at 13 months of age. The ferret subsequently showed decreasing motor activity and recumbency and was euthanized at 14 months of age. At necropsy, a white, quail egg-sized mass was found in the mesentery. Histopathologically, multifocal granulomas consisting of necrotic foci, macrophages, fibroblasts and plentiful fibrous connective tissues were observed in the mesenteric mass. Surrounding the granulomas, inflammatory cell infiltration consisting of neutrophils, lymphocytes and plasmacytes was observed diffusely and significantly. Immunohistochemistry revealed small numbers of macrophages around necrotic foci that were positively stained for anti-mouse feline coronavirus. Electron microscopically, the cytoplasm of the macrophages contained viral particles, which were identified as coronavirus. The histopathological features in this ferret were similar to those in cats with feline infectious peritonitis (FIP). This was the first case in ferrets in Japan.

Changes in plasma concentrations of corticosterone and its precursors after ketoconazole administration in rats: An application of simultaneous measurement of multiple steroids using LC-MS/MS.

The adrenal gland is the most common toxicological target in the endocrine system, and inhibition of adrenal steroidogenesis by drugs can be fatal in humans. However, methods to evaluate the drug effect are limited. Recently, simultaneous measurement of multiple steroids, including precursors, has become possible. Here, we evaluated the usefulness of this simultaneous measurement for the evaluation of drug effects on adrenal steroidogenesis in vivo. For this purpose, we measured plasma concentrations of adrenal steroids in rats dosed with ketoconazole, a known inhibitor of adrenal steroidogenesis, and examined its relationship with the changes in histopathology and mRNA expression of steroidogenic enzymes in the adrenal gland. Ketoconazole (150mg/kg/day) was orally administered to male rats for 7 days. The adrenal weight was high, and the zona fasciculata/reticularis were hypertrophic with an accumulation of lipid droplets. mRNA expression of CYP11A1, a rate-limiting enzyme in adrenal steroidogenesis, was slightly high in the adrenal gland. Plasma concentration of deoxycorticosterone was markedly high, while there were no significant changes in that of corticosterone, progesterone, or pregnenolone. The changes in the adrenal gland and plasma concentration of steroids were thought to reflect inhibited metabolism of deoxycorticosterone to corticosterone through inhibition of CYP11B1, and compensatory reaction for the inhibition. The compensatory reaction was thought to have masked decrease of corticosterone. These results suggest that simultaneous measurement of multiple steroids can enable sensitive evaluation of drug effects on adrenal steroidogenesis in vivo, while providing insight into the underlying mechanism of the effect.

Glomerulonephritis in a ferret with feline coronavirus infection

A male domestic ferret (Mustela putorius furo), which was purchased from outside of Japan at 13 weeks of age, was euthanized at 18 months of age because of poor health. At autopsy, the liver, spleen, and mesenteric lymph node were enlarged, and white foci were observed on the outer surface of the liver. The outer surface of the mesenteric lymph node was dark red. Histologically, granulomas were observed in the liver, spleen, bone marrow, and lymph nodes, composed mainly of aggregated epithelioid macrophages, some of which were positive to an anti–feline coronavirus (FCoV; Alphacoronavirus 1) antibody in immunohistochemistry. Mesangioproliferative glomerulonephritis was observed, and periodic acid–Schiff-positive deposits were observed along glomerular capillary walls. These deposits stained pale red with periodic acid–methenamine silver stain and red with Masson trichrome stain, and were also observed in the mesangial matrix. In affected glomeruli, glomerular capillary walls and mesangial areas were positive for anti-ferret immunoglobulin G. By electron microscopy, subepithelial and mesangial electron-dense deposits were observed consistent with immune complex deposition. The deposition of immune complexes may have been associated with FCoV infection.

Spontaneous Rhabdomyosarcoma in a Common Marmoset (Callithrix jacchus)

The common marmoset (Callithrix jacchus) is now widely used in various research fields, including toxicology. However, information about the background pathology of this species is scarce. Here, we report a case of rhabdomyosarcoma that spontaneously occurred in a common marmoset. A 44-month-old male common marmoset was euthanized due to bilateral hind limb paralysis. At necropsy, a 2×2×5-cm intramuscular mass was observed in the lower right back. Histologically, the mass was mainly composed of interlacing bundles of spindle-shaped tumor cells. Immunohistochemically, the tumor cells were positive for myogenin, desmin, vimentin and alpha-smooth muscle actin. Ultrastructurally, the tumor cells contained bundles of myofilaments with Z-band-like structures. Thus, the tumor was diagnosed as a rhabdomyosarcoma. To our knowledge, this is the first report of spontaneous rhabdomyosarcoma that was definitely diagnosed in the common marmoset.

Hyaline Glomerulopathy with Tubulo-Fibrillary Deposits in Young ddY Mice

Hyaline glomerulopathy with tubulo-fibrillary deposits was observed in two young female ddY mice. One of the mice showed gross systemic edema and bilateral enlargement and pale color of the kidneys, whereas no significant gross findings were noted in the other mouse. Microscopically, a large number of the glomeruli in both mice were enlarged because of diffuse and global deposition of amorphous eosinophilic materials. The deposits were negatively stained with Congo red and positively stained with IgG, IgM, IgA, C3, and periodic acid–Schiff. Electron microscopic examination revealed microtubular and fibrillary deposits with diameters of 80–100 and 9–16 nm, respectively, in the subendothelial space of the glomeruli. These features are histopathologically similar to immunotactoid glomerulopathy or fibrillary glomerulonephritis according to the classification of human glomerular lesions. Understanding of these characteristics of hyaline glomerulopathy in ddY mice is essential when evaluating pharmacological, pharmacokinetic, and toxicological studies using this mouse strain.

Establishment and Characterization of Renal Carcinoma Cell Lines from a Bhd Gene Mutant (Nihon) Rat

A germline insertion of a single nucleotide in the rat homologue of the human Birt-Hogg-Dubé (BHD) gene gives rise to dominantly inherited renal cell carcinoma (RCC) in the Nihon rat model. In this study, we established 7 lines (NR cell lines NR22, 24, 32, 45, 49, 54 and 64) from an RCC found in a Nihon rat. All cell lines consisted mainly of round or polygonal cells arranged in a cobblestone-like growth pattern. Cells of NR cell lines had abundant cytoplasm and tight junctions as well as microvilli on electron microscopy and were positive for cytokeratin on immunocytochemistry. Cell lines NR22, 24 and 32 showed rapid growth, whereas the growth of the remaining lines was very slow. While the modal chromosome number of lines NR24, 45 and 54 was 42, the remaining lines exhibited aberrant modal numbers ranging from 70 to 96. All NR cell lines formed tumors at subcutaneous inoculation sites in nude mice, and tumors from lines NR54 and 64 developed pulmonary metastases. All NR cell lines had a germline mutation in the rat Bhd gene in the gene analysis. NR cell lines would prove valuable experimental tools for studies on unique functions of the BHD gene and renal carcinogenesis.