Tsutomu Kurosawa

HEPATOCYTE GROWTH FACTOR PREVENTS RENAL FIBROSIS AND DYSFUNCTION IN A MOUSE MODEL OF CHRONIC RENAL DISEASE

Chronic renal disease (CRD) is generally thought to be incurable, except through renal transplantation, and the number of patients with CRD is on the increase. Glomerulosclerosis and tubulointerstitial fibrosis represent the morphological equivalent of end-stage CRD. In this study, we demonstrated the preventive effect of hepatocyte growth factor (HGF) on the progression of renal dysfunction and fibrosis, using a spontaneous mouse model for CRD (ICGN strain). The mice progressively developed glomerular sclerotic injury, tubular atrophy, and renal dysfunction until they were 17 wk of age. When recombinant HGF was injected into these mice during a 4-wk-period (from weeks 14-17 after birth), DNA synthesis of tubular epithelial cells was found to be 4.4-fold higher than in mice without HGF injection, thereby suggesting tubular parenchymal expansion promoted by HGF. Notably, HGF suppressed the expression of transforming growth factor-beta and of platelet-derived growth factor as well as myofibroblast formation in the affected kidney. Consequently, the onset of tubulointerstitial fibrosis was almost completely inhibited by HGF, while HGF attenuated the progression of glomerulosclerosis, both leading to preventing manifestation of renal dysfunction. From our results, supplement therapy with HGF may be taken into consideration as a novel option for prevention and treatment of CRD.

Hepatocyte growth factor (HGF) promotes oligodendrocyte progenitor cell proliferation and inhibits its differentiation during postnatal development in the rat

Hepatocyte growth factor (HGF) was initially cloned as a mitogen for hepatocytes and has been identified as a neurotrophic factor for a variety of neurons. However, few attempts have assessed the role of HGF in cells of oligodendrocyte lineage. The purpose of this study was to elucidate the role of HGF in such cells during development. Double immunostaining for either c-Met/HGF receptor or phospho-c-Met with either NG2 or RIP in rat striatum at postnatal day 3 (P3), P7, and P14 revealed that c-Met was phosphorylated on tyrosine residues and thereby activated in NG2(+) oligodendrocyte progenitor cells (OPCs) at P3-P14 and in RIP(+) oligodendrocytes at P14. Intrastriatal injections of recombinant human HGF at both P7 and P10 revealed that the relative ratio of BrdU(+)/NG2(+) cells per total number of NG2(+) cells increased, while BrdU(+)/MBP(+) oligodendrocyte numbers decreased. Western blot analysis showed a down-regulation of myelin basic protein (MBP) after HGF injection. Electron microscopy revealed that the numbers of myelinated nerve fibers decreased after HGF treatment. Furthermore, administration of anti-HGF IgG into the striatum increased the number of BrdU(+)/MBP(+) oligodendrocytes. These findings demonstrated that HGF increases proliferation of OPCs and attenuates their differentiation into myelinating oligodendrocytes, presumably by favoring neurite outgrowth that may be inhibited by the myelin inhibitory molecules on oligodendrocytes. Down-regulation of HGF mRNA in the striatum from P7 to P14, as revealed by quantitative real-time RT-PCR, may be favorable for OPC differentiation into myelinating oligodendrocytes. Our findings suggest that c-Met signaling, together with HGF regulation, plays an important role in developmental oligodendrogenesis.

Phylogenetic relationships of three hymenolepidid species inferred from nuclear ribosomal and mitochondrial DNA sequences

Three hymenolepidid tapeworms, Hymenolepis diminuta, H. nana and H. microstoma, are commonly maintained in laboratory rodents and used in many experimental model systems of tapeworm infections. We examined partial sequences from the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences to infer phylogenetic relationships of the 3 hymenolepidid species. Parts of the CO1 gene and ITS2 were amplified by PCR and sequenced directly. The CO1 gene sequence obtained was the same in length (391 bp) among all specimens. In the case of ITS2, however, several insertions and deletions were detected (671-741 bp) not only among species but also between an American isolate and a Japanese isolate of H. diminuta. Percentage nucleotide differences between H. diminuta and H. microstoma, or H. diminuta and H. nana were 16.6-18.2% for the CO1 gene and 21.3-22.9% for ITS2. The differences in both sequences between H. microstoma and H. nana were about 14%. Phylogenetic trees inferred from both of the nucleotide sequences showed similar topology, and suggest that H. diminuta may have diverged from the common ancestral line the earliest, and that H. nana is closer to H. microstoma than to H. diminuta.

Deficiency of the tensin2 gene in the ICGN mouse : an animal model for congenital nephrotic syndrome

The ICGN mouse is a model for nephrotic syndrome (NS) which presents with proteinuria, hyperlipidemia, and edema. In this study we attempted to identify the gene(s) responsible for NS. By analyzing albuminuria in 160 (ICGN × MSM)F1 × ICGN backcross progenies, we found that NS in the ICGN mouse is caused by more than one gene. We then performed a quantitative trait locus (QTL) analysis and detected a QTL with a very high LOD score peak in the telomeric region of Chr 15. By analyzing the nucleotide sequence of 22 genes located close to the QTL, we found that the tensin2 gene of the ICGN mouse possessed an 8-nucleotide deletion mutation in exon 18, leading to a frameshift and giving rise to a terminal codon at a premature position. Analyses of in situ hybridization and immunohistochemistry revealed that tensin2 was expressed in podocytes and tubular epithelial cells in normal mice but not in the ICGN mouse. These data raise the possibility that a mutation of the tensin2 gene is responsible for NS of the ICGN mouse and tensin2 is a prerequisite for the normal kidney function.

Anesthetic Effects of a Mixture of Medetomidine, Midazolam and Butorphanol in Two Strains of Mice

The combination of ketamine and xylazine is a widely used anesthetic for laboratory animals. However, due to an abuse problem in Japan, ketamine has been specified as a narcotic since 2007. Instead of using ketamine, Kawai et al. reported an injectable formula with an equivalent effect to the mixture of ketamine and xylazine [11]. The mixture of 0.3 mg/kg body weight (b.w.) medetomidine (Med.), 4.0 mg/kg b.w. midazoram (Mid.), and 5.0 mg/kg b.w. butorphanol (But.) produced an anesthetic duration of around 40 min in outbred ICR mice. However, the anesthetic effect of the mixture for inbred mice strains remains unknown. Therefore, we examined anesthetic effects of the mixture of Med., Mid., and But. in the BALB/c and C57BL/6J strains. After intraperitoneal injection into mice, right front paw, left hind paw, and tail pinch reflexes as well as corneal and righting reflexes were observed. Every 5 min, we scored each reflex category as 0 for reaction or 1 for no reaction. As long as the total score was at least 4 out of 5, we considered the mixture as putting a mouse in a surgical anesthetic state. The mixture produced an anesthetic duration of more than 45 min in both strains of mice. These results indicate that the mixture of Med., Mid., and But. can be a useful and effective anesthesia for the BALB/c and C57BL/6J strains of inbred mice as well as outbred ICR mice.

Nephrotic mice (ICGN strain) : A model of diffuse mesangial sclerosis in infantile nephrotic syndrome

The ICGN mouse strain is a unique model for naturally occurring nephrotic syndrome. In the present study, we examined the onset of the clinical manifestation of nephrotic syndrome and determined the sequence of intraglomerular events associated with progression of nephrotic conditions. Laboratory analysis revealed that homozygous (nep/nep) mice showed urinary albumin excretion during the suckling stage, rapidly leading to hypoalbuminemia accompanied by body growth failure. Renal pathology demonstrated that an initial intraglomerular event in the nephrotic mice was observed 3 weeks after birth in the form of mesangiolytic lesions, characterized by microaneurysm, platelet accumulation and capillary ballooning. In 6-week-old homozygous mice, mesangial sclerosis, characterized by mesangial expansion and glomerular hypertrophy, was observed in a diffuse fashion. Immunohistochemistry revealed that the glomerular cells in the 3-week-old homozygous suckling mice were positive for α-smooth muscle actin, suggesting a phenotypic change in the mesangial cells. Mesangial expansion, confirmed by the over-deposition of type I collagen, was evident until 6 weeks after weaning, while it was of interest that fibrogenic cytokines such as platelet-derived growth factor and transforming growth factor-β were not detected in the sclerotic glomeruli throughout the observations. Furthermore, the nephrotic features were shown to be resistant to steroid therapy with a high dose of prednisolone. Our results suggest that diffuse mesangial sclerosis, a hereditary glomerular disease, may be genetically generated through early myofibroblast formation, which occurs and develops probably independently of up-regulation of these fibrogenic cytokines. In conclusion, the homozygous nephrotic mouse (ICGN strain) is believed to be a good model for investigating not only nephrotic conditions but also cellular and molecular pathogenesis of diffuse mesangial sclerosis in steroid-resistant infantile nephrotic syndrome.

N-Glycosylation modulates the membrane sub-domain distribution and activity of glucose transporter 2 in pancreatic beta cells.

The glucose transporter isoform, GLUT2, -mediated glucose sensing is essential for maintaining normal glucose-stimulated insulin secretion in pancreatic beta cells. We previously reported that GnT-IVa glycosyltransferase is required for the production of an N-glycan structure that acts as a ligand for galectins to form the glycan-galectin lattice that maintains the stable cell surface expression of GLUT2, and cellular glucose transport activity, although the functional relevance of the N-glycosylation of GLUT2 to its membrane sub-domain distribution is not fully understood. In the present study, we demonstrated that disruption of the GLUT2 N-glycan-galectin lattice by the genetic inactivation of GnT-IVa, or by treatment of pancreatic beta cells with competitive glycan mimetics, induced the re-distribution of GLUT2 into the lipid-raft microdomain. This subsequently resulted in the binding of Stomatin to GLUT2 and an attenuation of cellular glucose transport activity. Moreover, disruption of the lipid-raft microdomain by treatment with methyl-β-cyclodextrin caused the GLUT2 to be released from lipid-rafts and reactivation of the cellular glucose transport activity in GnT-IVa deficient beta cells. These results indicate that the membrane sub-domain distribution of GLUT2 is associated with the glucose transport activity of beta cells, in which the GnT-IVa-dependent formation of the N-glycan-galectin lattice plays an important role. This provides a novel pathophysiological insight into the mechanism of beta cell failure in the pathogenesis of type 2 diabetes.

Intraspecific variation of isoenzymes in Taenia taeniaeformis.

The technique of isoenzyme electrophoresis was applied to Japanese wild populations of Taenia taeniaeformis (isolated from Norway rats) and three laboratory reared isolates (KRN isolated from a Malaysian Norway rat, BMM from a Belgian house mouse and ACR from a Japanese gray red-backed vole). The average heterozygosities of Japanese wild populations were fairly small and total genetic variability was 0.0499. The genetic make-up of T. taeniaeformis in Norway rats was rather uniform in the whole of Japan. In KRN isolate, each of all 10 loci examined possessed the allele which was predominant in Japanese wild populations. Similarly, each of 9 loci in BMM isolate possessed the same alleles, but one of 2 alleles at HK locus was different from that in the others. T. taeniaeformis parasitizing house mice and rats were considered to be genetically closely related to each other. In ACR isolate, 7 out of 10 loci possessed different alleles from those in the other populations. It was considered that ACR isolate was genetically distant and its phylogenetic origin in Japan should be different from worms parasitizing Norway rats.

Preventive effect of ACE inhibitor on interstitial myofibroblast formation and matrix deposition in a nephrotic model

The nephrotic mouse (ICGN strain) is a useful model for progressive nephrotic syndrome (NS). In the present study, we demonstrated the preventive effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor, on the progression of renal dysfunction and tubulo-interstitial fibrosis in the NS mice. Administration of enalapril (5 mg/dL in drinking water) to the 4-week-old NS mice for a 4-week-period did not improve their nephrotic symptoms such as albuminuria and hypoalbuminemia, but significantly suppressed the increases in blood urea nitrogen and serum creatinine levels. Renal histopathology demonstrated that the administration of the ACE inhibitor significantly attenuated the progression of the tubular and interstitial lesions (tubular dilatation, luminal cast accumulation and interstitial expansion) rather than the glomerular sclerotic changes. The suppression of the increase in blood urea nitrogen level by enalapril depended on the attenuated tubular injury rather than on the unchanged glomerular matrix deposition. Immunohistochemical examination revealed that the administration of the ACE inhibitor suppressed the formation of myofibroblasts, identified by the alpha-smooth muscle actin-positive cells, in the interstitial spaces. Consequently, interstitial matrix deposition was significantly reduced in the NS mice treated with enalapril. From the results obtained with the spontaneous nephrotic model, we emphasize a possibility that ACE inhibitor may be effective for attenuating progression of renal dysfunction and fibrosis in human NS, even if the ACE inhibitor fails to improve nephrotic symptoms such as albuminuria and hypoalbuminemia.

Invention of a forced-air-ventilated micro-isolation cage and rack system Environment within cages: temperature and ammonia concentration

Abstract Isolator caging systems provide individual animal microenvironments and can be useful in the research setting. This caging system may be required to provide a barrier for specific-pathogen-free rodents at the cage level. In this study, we designed a forced-air ventilation system (FVMIS) and evaluated its ability to control such environmental factors as temperature and ammonia concentration within cages. We found that the FVMIS was capable of controlling the temperature uniformly and accurately while housing rats. In addition, microenvironmental ammonia concentrations, determined daily from days 1 through 16, showed a delayed rise in ammonia concentration by at least 4 days in FVMIS cages compared to non-ventilated control cages. This suppression of ammonia allows for a reduction in the frequency of cage changes.