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camera (DS-L3; Nikon, Tokyo, Japan) at 400× magnification. To determine any changes in the number of nephrons, we counted the glomeruli and immature glomeruli in the whole section starting from the central region of the kidney across the full coronal plane in P19 pups. Mature glomeruli were defined as glomeruli with loose tuft structure, lobu-lation, and patent capillary loops lined with typical podocytes, whereas immature glomeruli referred to glomeruli with at least half of the circumference of capillary loops was densely lined with dark cuboidal epithelial cells. The lumen of the loops is generally narrow, with no tuft lobulation.Reverse transcription-polymerase chain reaction (RT-PCR)The expression levels of α-SMA, TGF-β, and TNF-α in the renal cortex were determined by real-time RT-PCR using the TaqMan system according to the manufacturer’s protocol. The samples were homogenized with a buffer RLT added and rotated twice at 3000 rpm for 30 seconds using a rotor-stator homogenizer. TaqMan probe-based quantitative RT-PCR was conducted using cDNA synthesized from kidney biopsy. RNA was prepared using the High-Capacity cDNA Reverse Transcription Kit and analyzed using the default Figure 1a　Histological images of the kidneys of newborn ratsIn the O2-PW group, the number of immature glomeruli significantly increased (Hematoxylin and eosin staining of 3μm-thick kidney sections; 100× and 400× magnification).Abbreviations: RA-PW, the group reared in room air with purified water; RA-HW, the group reared in room air with hydrogen-rich water; O2-PW, the group reared in 80% oxygen with purified water; O2-HW, the group reared in 80% oxygen with hydrogen-rich water.protocols of the 7500 Fast Real-Time PCR System (Life Technologies). Using the Standard Curve Method, we normalized each gene expression to GAPDH gene expression. Furthermore, primers and probes for α-SMA ACTA (Rn01759928_g1), TGF-β (Rn00572010_m1), and TNF-α (Rn99999017_ m1) were prepared using TaqMan Gene Expres-sion Assays.Statistical analysisData are expressed as mean ± standard devia-tion. We used one-way analysis of variance to determine the differences between groups and the Bonferroni method for post hoc multiple compari-sons. All statistical data were analyzed using SPSS Statistics software, version 17.0 (SPSS Inc., Chicago, IL, USA), and a P value of < 0.05 was considered statistically significant.Effect of hyperoxia on glomeruliHyperoxia exposure did not change the body weight, kidney weight, and the number of glom-eruli per section. However, the number of imma-ture glomeruli in the O2-PW group significantly increased (Figure 1a). The average number of immature glomeruli in the RA-PW and O2-PW 237Results