Acceleration of Age-Related Changes in the Retina in ␣-Tocopherol Transfer Protein Null Mice Fed a Vitamin E-Deficient Diet

PURPOSE. To assess the role of vitamin E (VE) in age-related changes in the retinal tissues by using a mouse model of severe VE deficiency. METHODS. Pups of ␣-tocopherol transfer protein null (a-TTP Ϫ/Ϫ ) mice were fed a VE-deficient diet for 4 or 18 months (VE (Ϫ) group). Wild-type C57BL/6 mice were fed a 0.002% ␣-tocopherol-supplemented diet (VE (ϩ) group). In various ocular tissues, the VE levels were measured by high-performance liquid chromatography; the fatty acid composition by gas chromatography (GC); and the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F 2␣ levels, which are biomarkers for lipid peroxidation, by GC-mass spectrometry. The retinal structure was assessed by light, electron, and fluorescence microscopy. RESULTS. The ␣-tocopherol level in the retinas obtained from 4-month-old VE (Ϫ) animals was 71-fold lower than that in the retinas obtained from the VE (ϩ) group. In addition, ␥-tocopherol was not detected; thus, the VE (Ϫ) group demonstrated a more severe VE deficiency than ever reported. In this group, the concentration of n-3 polyunsaturated fatty acids decreased (0.3-to 0.9-fold), whereas that of other classes of fatty acids was unchanged or increased. At 18 months of age, the number of the outer nuclear layer (ONL) nuclei was observed to be 17% lower in the VE (Ϫ) than in the VE (ϩ) group (P Ͻ 0.05). Electron microscopy revealed larger amounts of matrix between the ONL nuclei indicating the Müller cell hypertrophy, greatly expanded rod outer segment discs, and a larger number of inclusion bodies in the retinal pigment epithelium (RPE; P Ͻ 0.05) in the VE (Ϫ) group. Fluorescence microscopy revealed that the autofluorescence signal was increased in the RPE layer in this group. When the observations of the 18-month-old animals were compared to those of the 4-month-old animals, the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F 2␣ levels were found to be increased in the retina and RPE obtained from both the VE (Ϫ) and VE (ϩ) groups; however, the age-related increases were more remarkable in the VE (Ϫ) group (2.6-to 43.5-fold) than in the VE (ϩ) group (0.8-to 8.7-fold). CONCLUSIONS. The combined use of a-TTP Ϫ/Ϫ mice and a VEdeficient diet leads to a severe deficiency of VE, enhances lipid peroxidation in the retina, and accelerates degenerative damage of the retina with age. (Invest Ophthalmol Vis Sci. 2007; 48:396 -404

SGLT5 Reabsorbs Fructose in the Kidney but Its Deficiency Paradoxically Exacerbates Hepatic Steatosis Induced by Fructose

Although excessive fructose intake is epidemiologically linked with dyslipidemia, obesity, and diabetes, the mechanisms regulating plasma fructose are not well known. Cells transfected with sodium/glucose cotransporter 5 (SGLT5), which is expressed exclusively in the kidney, transport fructose in vitro; however, the physiological role of this transporter in fructose metabolism remains unclear. To determine whether SGLT5 functions as a fructose transporter in vivo, we established a line of mice lacking the gene encoding SGLT5. Sodium-dependent fructose uptake disappeared in renal brush border membrane vesicles from SGLT5-deficient mice, and the increased urinary fructose in SGLT5-deficient mice indicated that SGLT5 was the major fructose reabsorption transporter in the kidney. From this, we hypothesized that urinary fructose excretion induced by SGLT5 deficiency would ameliorate fructose-induced hepatic steatosis. To test this hypothesis we compared SGLT5-deficient mice with wild-type mice under conditions of long-term fructose consumption. Paradoxically, however, fructose-induced hepatic steatosis was exacerbated in the SGLT5-deficient mice, and the massive urinary fructose excretion was accompanied by reduced levels of plasma triglycerides and epididymal fat but fasting hyperinsulinemia compared with fructose-fed wild-type mice. There was no difference in food consumption, water intake, or plasma fructose between the two types of mice. No compensatory effect by other transporters reportedly involved in fructose uptake in the liver and kidney were indicated at the mRNA level. These surprising findings indicated a previously unrecognized link through SGLT5 between renal fructose reabsorption and hepatic lipid metabolism

BMP4の機能調節は糖尿病性腎症及びポドサイト障害の治療につながる

Podocyte injury has been proposed to play an important role in diabetic nephropathy; however, its pathological mechanism remains unclear. We have shown that bone morphogenetic protein 4 (BMP4) signaling leads to the glomerular changes characteristic of this disorder. To analyze the molecular mechanism of podocyte injury, the effect of BMP4 was investigated using streptozotocin (STZ)- induced, Bmp4 heterozygous knockout (Bmp4+/−) and podocyte-specific Bmp4 knockout mice. Mice with STZ-induced diabetes exhibited glomerular matrix hyperplasia and decreased numbers of podocyte nucleus-specific WT1-positive cells. The number of podocytes and proteinuria were improved in both diabetic Bmp4 knockout mouse models compared to the effects observed in the control mice. The effect of BMP4 overexpression on Bmp4-induced or podocyte-specific transgenic mice was examined. Tamoxifen-induced Bmp4-overexpressing mice exhibited mesangial matrix expansion and decreased numbers of WT1-positive cells. Podocyte-specific Bmp4-overexpressing mice displayed increased kidney BMP4 expression and mesangial matrix expansion but decreased nephrin expression and numbers of WT1-positive cells. Both lines of Bmp4-overexpressing mice exhibited increased albuminuria. In cultured podocytes, BMP4 increased phospho-p38 levels. BMP4 decreased nephrin expression but increased cleaved caspase-3 levels. p38 suppression inhibited caspase-3 activation. Apoptosis was confirmed in STZ-diabetic glomeruli and Bmp4-overexpressing mice. Bmp4 +/− mice with diabetes displayed reduced apoptosis. Based on these data, the BMP4 signaling pathway plays important roles in the development of both podocyte injury and mesangial matrix expansion in diabetic nephropathy

Conditional deletion of Npt2b in phosphate transport

Background Hyperphosphatemia is common in chronic kidney disease and is associated with morbidity and mortality. The intestinal Na+-dependent phosphate transporter Npt2b is thought to be an important molecular target for the prevention of hyperphosphatemia. The role of Npt2b in the net absorption of inorganic phosphate (Pi), however, is controversial. Methods In the present study, we made tamoxifen-inducible Npt2b conditional knockout (CKO) mice to analyze systemic Pi metabolism, including intestinal Pi absorption. Results Although the Na+-dependent Pi transport in brush-border membrane vesicle uptake levels were significantly decreased in the distal intestine of Npt2b CKO mice compared with control mice, plasma Pi and fecal Pi excretion levels were not significantly different. Data obtained using the intestinal loop technique showed that Pi uptake in Npt2b CKO mice was not affected at a Pi concentration of 4 mM, which is considered the typical luminal Pi concentration after meals in mice. Claudin, which may be involved in paracellular pathways, as well as claudin-2, 12, and 15 protein levels were significantly decreased in the Npt2b CKO mice. Thus, Npt2b deficiency did not affect Pi absorption within the range of Pi concentrations that normally occurs after meals. Conclusion These findings indicate that abnormal Pi metabolism may also be involved in tight junction molecules such as Cldns that are affected by Npt2b deficiency

Transportation of Freeze-Dried Mouse Spermatozoa Under Different Preservation Conditions

Freeze-dried mouse spermatozoa can be used for normal embryonic development after injection into oocytes, thus indicating that freeze-drying is a useful method for the storage and transportation of genetic materials from animals. We recently reported that storage of freeze-dried mouse spermatozoa requires maintenance at temperatures lower than -80 C for long-term preservation and a pressure of 0.37 mbar at primary drying and that these conditions significantly improve the developmental rate to the blastocyst stage. In this study, we examined the influence of transportation and preservation conditions on freeze-dried spermatozoa. Freeze-dried spermatozoa stored for 2 or 2.5 years at 4 or -80 C were transported round trip overland between Shizuoka and Hokkaido prefectures in Japan or by air between Japan and Belgium. The freeze-drying conditions consisted of primary drying at pressures of 0.04, 0.37 and 1.03 mbar and secondary drying at a pressure of 0.001 mbar. Embryos (2-cell stage) from freeze-dried spermatozoa dried at 0.04 mbar and stored at 4 C for 2 years with and without overland transportation did not develop to term. The development rates of embryos from spermatozoa stored at -80 C for up to 2 years and transported overland, by air and without transportation were 8, 1 and 28%, respectively. The development rates of embryos from spermatozoa without transportation were significantly higher than with transportation (P<0.05). These data indicate that freeze-dried spermatozoa stored at -80 C with and without transportation can retain their ability to generate viable offspring after storage for up to 2 years. However, there are limitations to be considered in the transportation of freeze-dried spermatozoa at ambient temperature.http://www.jstage.jst.go.jp/article/jrd/53/6/53_1169/_article/-char/ja

Effect of Zona Incision by Piezo-Micromanipulator (ZIP) on In Vitro Fertilization in 21 Transgenic Mice Lines

Zona incision using a piezo-micromanipulator (ZIP) has been demonstrated to be effective for in vitro fertilization (IVF) using cryopreserved C57BL/6 spermatozoa. In this study, ZIP oocytes inseminated with frozen-thawed genetically modified C57BL/6J or FVB mice spermatozoa (21 lines) showed fertilization rates of 22-75% and live fetus rates of 8-49%. In 6 of the lines, the fertilization rates for oocytes compared with ZIP (42-75%) were significantly higher than that of nontreated oocytes (0-50%). Using only 90 oocytes for IVF with ZIP, 5 breeding pairs were produced from cryopreserved genetically modified mice spermatozoa. Our results indicate that application of the ZIP technique is effective for IVF using cryopreserved genetically modified mouse spermatozoa.http://www.jstage.jst.go.jp/article/expanim/58/4/58_415/_article/-char/ja

Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons

Formation of highly organized neocortical structure depends on the production and correct placement of the appropriate number and types of neurons. POU homeodomain proteins Brn-1 and Brn-2 are coexpressed in the developing neocortex, both in the late precursor cells and in the migrating neurons. Here we show that double disruption of both Brn-1 and Brn-2 genes in mice leads to abnormal formation of the neocortex with dramatically reduced production of layer IV–II neurons and defective migration of neurons unable to express mDab1. These data indicate that Brn-1 and Brn-2 share roles in the production and positioning of neocortical neuron development

The Role Of Salivary Glands In Phosphate Homeostasis

Hyperphosphatemia is recognized as a contributor to vascular calcification in patients with chronic kidney disease (CKD) and hemodialysis (HD) patients and is independently associated with cardiac mortality. Dietary inorganic phosphorus (Pi) restriction, and the Pi binders are important therapy for dialysis patients with hyperphosphatemia. Recent study reported that salivary secretion of Pi to be an important determinant of hyperphosphatemia in patients with CKD and in those with ESRD under chronic dialysis. In the present study, we investigated the role of type IIb sodium-dependent Pi transporter (Npt2b) on salivary Pi excretion in mice. The expression of Npt2b protein was detected at the apical side of duct cells in the salivary glands, suggesting that ductal cells appears to be able to reabsorb Pi, thereby modifying the Pi concentration in the final saliva. In wild-type mice (Wt mice) fed a high Pi diet, the levels of plasma and salivary Pi are significantly higher than those in mice fed a low Pi diet. In Npt2b+/- mice, the salivary Pi concentrations were significantly increased compared with those in Npt2b+/+ mice. Npt2b+/- mice with adenine-induced renal failure had low plasma and salivary Pi levels, and plasma creatinine and BUN levels compared with Npt2b+/+ mice treated with adenine. In conclusion, Npt2b is involved in Pi secretion by salivary glands