熊本 海生航

The live attenuated human rotavirus vaccine strain RIX4414 (Rotarix®) is used worldwide to prevent severe rotavirus-induced diarrhea in infants. This strain was attenuated through the cell culture passaging of its predecessor, human strain 89-12, which resulted in multiple genomic mutations. However, the specific molecular reasons underlying its attenuation have remained elusive, primarily due to the absence of a suitable reverse genetics system enabling precise genetic manipulations. Therefore, we first completed the sequencing of its genome and then developed a reverse genetics system for the authentic RIX4414 virus. Our experimental results demonstrate that the rescued recombinant RIX4414 virus exhibits biological characteristics similar to those of the parental RIX4414 virus, both in vitro and in vivo. This novel reverse genetics system provides a powerful tool for investigating the molecular basis of RIX4414 attenuation and may facilitate the rational design of safer and more effective human rotavirus vaccines.

A long-term high-fat diet (HFD) causes obesity and changes in renal lipid metabolism and lysosomal dysfunction in mice, causing renal damage. Sodium-glucose co-transporter inhibitors, including phlorizin, exert nephroprotective effects in patients with chronic kidney disease, but the underlying mechanism remains unclear. A HFD or standard diet was fed to adult C57BL/6J male mice, and phlorizin was administered. Lamellar body components of the proximal tubular epithelial cells (PTECs) were investigated. After phlorizin administration in HFD-fed mice, sphingomyelin and ceramide in urine and tissues were assessed and label-free quantitative proteomics was performed using kidney tissue samples. Mitochondrial elongation by fusion was effective in the PTECs of HFD-fed obese mice under phlorizin administration, and many lamellar bodies were found in the apical portion of the S2 segment of the proximal tubule. Phlorizin functioned as a diuretic, releasing lamellar bodies from the apical membrane of PTECs and clearing the obstruction in nephrons. The main component of the lamellar bodies was sphingomyelin. On the first day of phlorizin administration in HFD-fed obese mice, the diuretic effect was increased, and more sphingomyelin was excreted through urine than in vehicle-treated mice. The expressions of three peroxisomal β-oxidation proteins involved in fatty acid metabolism were downregulated after phlorizin administration in the kidneys of HFD-fed mice. Fatty acid elongation protein levels increased with phlorizin administration, indicating an increase in long-chain fatty acids. Lamellar bodies accumulated in the proximal renal tubule of the S2 segment of the HFD-fed mice, indicating that the urinary excretion of lamellar bodies has nephroprotective effects.

BACKGROUND: Cystogenesis in polycystic kidney disease (PKD) is likely accelerated by various renal insults, including crystal deposition, that activate renal tubule obstruction and dilation. We developed a capsule-based device that can be applied to cystic kidneys to restrict tubular lumen dilatation and cyst expansion. METHODS: Kidney capsule devices were designed from computed tomography images of wild-type and Cy/+ rats. Capsule devices were surgically implanted on kidneys in six surgical sessions over a period of 14 months in 7 wild-type rats of 6.5-8 weeks (3 sham operations, 2 right, 2 left) and 6 Cy/+ rats of 6.5 weeks (2 sham, 3 left, 1 bilateral). After surgery, the rats were followed for 5.4-12.4 weeks' growth and sacrificed to retrieve the kidneys. During the follow-up, serum creatinine was measured and retrieved kidneys were weighed. Histological analysis including cystic area measurement and immunohistochemistry was performed. RESULTS: Morphometric capsule devices were configured and developed by an image processing technique and produced using a 3D printer. Encapsulated Cy/+ kidneys (n = 5; mean weight 3.64 g) were consistently smaller in size (by 21-36%; p < 0.001) than unencapsulated Cy/+ kidneys (n = 7; mean weight 5.52 g). Encapsulated Cy/+ kidneys (mean %cyst area: 29.4%) showed smaller histological cystic area (by 28-58%; p < 0.001) than unencapsulated Cy/+ kidneys (mean %cyst area 48.6%). Cell proliferation and macrophages were also markedly reduced in encapsulated Cy/+ kidneys, compared to unencapsulated Cy/+ kidneys. CONCLUSIONS: We report a pilot feasibility study for the application of a novel morphometric 3D capsule device to the Cy/+ rat model showing restricted kidney volume expansion on polycystic kidney disease progression.