Masaki Mino

Maternal protein restriction that does not have an influence on the birthweight of the offspring induces morphological changes in kidneys reminiscent of phenotypes exhibited by intrauterine growth retardation rats.

Severe restriction of maternal protein intake to 6–8% protein diet results in intrauterine growth retardation (IUGR), low birthweight and high risk of metabolic syndrome in the adult life of the offspring. However, little information is available on the effects of maternal protein restriction on offspring under the conditions that does not have an influence on their birthweight of the offspring,. In the present study, pregnant rats were kept on a diet consisting of either 9% (low‐protein, Lp rats) or 18% (normal‐protein, Np rats) protein by weight/volume/etc. After birth, both Lp and Np rats were kept on a diet containing 18% protein. Neonatal body weight was significantly lower in Lp rats compared to Np rats from 4 days to 5weeks after birth. While glomerular number per unit volume (1 mm3) of the kidney (Nv) was comparable between Lp and Np rats 4 weeks after birth, the Nv was significantly decreased in Lp rats at 20 weeks after birth. Four and 20 weeks after birth, glomerular sclerosis index, interstitial fibrosis score, and ratio of ED1‐positive cell ratio were all significantly higher in Lp compared to Np rats. Transforming growth factor‐β1‐positive cells were observed in the distal tubules in the kidney of 4‐ and 20‐week‐old Lp rats kidneys, but not in those of age‐matched Np rats. Altogether, these findings revealed that maternal protein restriction that does not have an influence on the birthweight of the offspring, induces similar changes as those seen in the kidneys of IUGR neonates.

Effects of Transient Forebrain Ischemia on the Hippocampus of the Mongolian Gerbil (Meriones unguiculatus): An Immunohistochemical Study

In the Mongolian gerbil, bilateral common carotid artery occlusion (BCCAO) for several minutes induces ischemia and delayed neuronal cell death in the CA1 region of the hippocampus due to their incomplete Circle of Willis. In the present study, the expression of fibroblast growth factor 2 (FGF2), its receptors (FGFR1 and FGFR2), glial fibrillary acidic protein (GFAP), and isolectin B4 (ISLB4) was investigated by immunohistochemical and lectin-binding methods after BCCAO was performed for 5 min in gerbils. One day after BCCAO, the pyramidal cells of the CA1 region of the hippocampus showed degenerative changes and lowered expression of FGF2, FGFR1, and FGFR2. Three days after BCCAO, there was an increase in GFAP-positive astrocytes and ISLB4-positive microglial cells. From five to 10 days after BCCAO, intense neuronal cell death in the stria pyramidale of the hippocampal CA1 region was observed, as well as an increase in GFAP-positive astrocytes and decrease in ISLB4-positive microglial cells. These results indicate that transient forebrain ischemia induces neuronal cell death with lowered expression of FGF2 and its receptors, and that the activation of glial cells may not directly lead to neuronal cell death.

Effects of low protein intake on the development of the remaining kidney in subtotally nephrectomized immature rats: expression of inducible and endothelial NO synthase

We examined the effects of low protein intake on the development of the remaining kidney in subtotally (5/6) nephrectomized immature rats. Three-week-old rats were kept on a diet containing either 12% protein (Lp rats) or 18% protein (Np rats) for 4 or 8 weeks after subtotal nephrectomy (SUNx). In Western blot analysis, the endothelial NO synthase (eNOS) protein expression of the Lp rats was significantly higher than that of the Np rats at 4 weeks after SUNx. Immunohistochemically, more inducible NO synthase (iNOS)-positive cells were observed in the Np rats than in the Lp rats 4 weeks after SUNx in the distal tubules. In semiquantitative RT-PCR, the expression of renin mRNA was significantly lower in the Lp rats than in the Np rats at 4 and 8 weeks after SUNx. These findings reveal that protein restriction is effective in preventing renal failure of immature rats and that the changes in the expression levels of renin, eNOS, and iNOS is involved in the process of this prevention.

An increase in apoptosis and reduction in αB-crystallin expression levels in the lens underlie the cataractogenesis of Morioka cataract (MCT) mice

We examined the morphological changes in fibers, localization of apoptotic cells, and protein expression of αB-crystallin in the lens of Morioka cataract (MCT) mice, a novel cataract model. Using a scanning electron microscope, swollen lens fibers and enlarged spaces between lens fibers were observed in the lens of 3-week-old MCT mice. At 2 weeks of age (before cataract), the single-strand DNA (ssDNA)-positive (indicating apoptosis) cell ratio of the lens epithelium was significantly higher in MCT than in wild-type ddY mice. At 2 and 4 weeks of age, αB-crystallin protein expression of the lens in MCT mice was significantly lower than that in wild-type ddY mice. These findings suggest that increase in apoptosis and reduction in αBcrystallin level are involved in the cataractogenesis of MCT mice.

Effects of maternal uninephrectomy on the development of fetal rat kidney: apoptosis and the expression of oncogenes.

ABSTRACT    The present study was designed to explore whether maternal uninephrectomy affects development of the collecting ducts in fetal kidney. Localization of DNA fragmented cells in the kidney of fetal rats from uninephrectomized mothers were examined by the terminal deoxynucleotidyl transferase (TdT)‐mediated d‐UTP‐biotin nick end labeling (TUNEL) method. Immunohistochemistry was used to examine the localizations of bcl‐2 gene products. The gene expressions for bcl‐2, p53, and WT1 mRNAs were examined by using the semi‐quantitative reverse transcript‐polymerase chain reaction. TUNEL positive cells were more numerous in the medullary collecting ducts of the fetuses from uninephrectomized mothers than in those of the fetuses from sham‐operated ones. The expressions of bcl‐2, p53, and WT1 mRNAs were lower in the fetuses from uninephrectomized mothers than in the fetuses from sham‐operated ones. Cells in the medullary collecting ducts showed positive reactions to anti‐bcl‐2 gene products antibody with the reactions being weaker in the fetuses from uninephrectomized mothers. These results showed that maternal uninephrectomy accelerated the development of fetal rat kidney and it was associated with the lowered the expression of bcl‐2 in fetal rat kidney.

Effects of maternal subtotal nephrectomy on the development of the fetal kidney: A morphometric study

The present study was designed to explore if maternal subtotal (5/6) nephrectomy affects the development of fetal rat kidneys using morphometric methods and examining whether there are any apoptotic changes in the fetal kidney. To generate 5/6 nephrectomized model rats, animals underwent 2/3 left nephrectomy on gestation day (GD) 5 and total right nephrectomy on GD 12. The fetal kidneys were examined on GDs 16 and 22. A significant decrease in fetal body weight resulting from maternal 5/6 nephrectomy was observed on GD 16, and a significant decrease in fetal renal weight and fetal body weight caused by maternal nephrectomy was observed on GD 22. Maternal 5/6 nephrectomy induced a significant increase in glomerular number, proximal tubular length, and total proximal tubular volume of fetuses on GD 22. Maternal 5/6 nephrectomy resulted in an increase in the number of apoptotic cells in the metanephric mesenchyme of the kidney on GD 16, and in the collecting tubules on GD 22. These findings suggest that maternal 5/6 nephrectomy stimulates the development of the fetal kidney while suppressing fetal growth.

Expression of transforming growth factor β and fibroblast growth factor 2 in the lens epithelium of Morioka cataract mice

In the Morioka cataract (MCT) mice, lens opacity appears at 6 to 8 weeks of age, and swollen lens fiber is electron‐microscopically observed at 3 weeks after birth. The present study was designed to characterize the expression of transforming growth factor β (TGFβ) and fibroblast growth factor 2 (FGF2) in the lens epithelium of the MCT mice. Immunohistochemical analysis showed that the expression of TGFβ in the lens epithelium of the MCT mice was stronger than that of the wild‐type ddY mice at 2 and 4 weeks after birth. The expression of TGFβ receptors (TGFβRI and TGFβRII) and FGF2 in the lens epithelium of the MCT mice was stronger than that of the wild‐type ddY mice at 4 weeks and weaker than that of the wild‐type ddY mice at 15 weeks after birth. Using real time polymerase chain reaction (PCR), quantitative RT‐PCR analysis showed that expression of TGFβ1 and TGFβ2 mRNA in the lens of 2‐week‐old MCT mice was significantly higher compared to age‐matched wild‐type ddY mice. These findings indicate that the lens epithelium of MCT mice has increased expression of TGFβ before cataract affection and that changes in the expression of FGF2 as well as TGFβ may contribute to the progression of the cataract in the mice.

Effects of Maternal Renal Dysfunction on Fetal Development

Maternal conditions affect the growth of the fetal kidney, which begins to secrete urine during late gestation (Bakala et al., 1985; Schaeverbeke & Cheignon, 1980). For example, during pregnancy, the maternal kidney undergoes various changes such as an increase in glomerular filtration rate (Baylis, 1994; Atherton & Pirie, 1981) and an alteration in tubular function (Dafnis & Sabatini, 1992). Furthermore, maternal undernutrition by the restriction of protein intake induces low fetal birth and leads to renal morphological and physiological changes (Mesquita et al., 2010). Maternal bilateral ligation of the uterine artery, protein restriction, smoking, nephrotoxic medication as well as salt loading cause intrauterine growth retardation (IUGR) to fetuses (Bentz & Amann, 2010), inducing fetal programing of renal function. Previously, we investigated the development of the fetal kidney during maternal renal dysfunction induced by bilateral ureteral ligation (Okada et al., 1997) and uninephrectomy (Okada et al., 2000, 2006), and we found that the development of the fetal kidney is accelerated by the operations. Furthermore, we found that maternal bilateral ureteral ligation (Okada & Morikawa, 1988) and uninephrectomy (Okada et al., 1998) decrease fetal body weight. In this chapter, we present a summary of the changes that occur in the fetal kidney after maternal bilateral ureteral ligation, uninephrectomy, and subtotal (5/6) nephrectomy, in addition to a summary of the changes that occur in the remaining kidney after uninephrectomy and 5/6 nephrectomy. Although 5/6 nephrectomy produces a bigger functional demand to maternal and fetal kidneys than the other two operations, little information is available on fetal development of the kidney when the maternal kidney is 5/6th removed. Therefore, we focused on studying the remaining kidney of unilateral and 5/6 nephrectomy and the fetal kidney under maternal renal dysfunction.