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(Shizuoka, Japan) and housed in individual cages in the same room at 24°C-25°C, with a relative humidity of 60% under a 12-hour/12-hour light/dark cycle and free access to food and water, at the Juntendo University Animal Care Facility. Pups were born naturally at term and maintained in either 80% oxygen (a mixture of medical-grade 100% oxygen and room air [RA]; Oxycycler ProOx 110; Biospherix, Lacona, NY, USA) or RA from postnatal day (PD) 0 to PD 12. The approval number obtained from the Juntendo University Animal Care Facility is 2020176.Experimental groupsPregnant Sprague-Dawley rats were divided into four groups. From birth to PD 12 , two groups were bred in 80% oxygen, and the other two were in RA. HW (O2-HW and RA-HW groups) or puri-fied water (PW; O2-PW and RA-PW groups) was administered to parents of each group after birth. To administer water and molecular hydrogen to newborn rats, there is a method of injecting water and molecular hydrogen directly into the stomach with a gastric catheter. However, this method had a problem in continuing the experiment for small rats. Therefore, with a view to future clinical appli-cation, we selected a method in which water and molecular hydrogen were orally administered to the mother rat and the effect was judged in the newborn rat raised by the mother rat’s breast milk. The pups were sacrificed at PD 19, and their kidneys were excised for tissue histologic examina-tions and biochemical assays. The weight of kidneys, number of glomeruli and immature glom-eruli, and levels of other markers were compared and validated.Histopathological and immunohistochemical analysesThe harvested kidneys were fixed in 10% formalin, embedded in paraffin, and sliced into sections starting from the central region of the kidney across the full coronal plane. The slides containing the sliced sections were stained with hematoxylin and eosin and examined by optical microscopy. For each section, three microphoto-graphs from the anterior, posterior, and mediolat-eral regions of the kidney, each including the full thickness of the cortex, were obtained using a digital 236as well as increased proportion of immature glom-eruli, are observed in this population. Thus, post-natal nephrogenesis may be potentially impaired3, 4). Several children and adults born prematurely suffer from hypertension5, 6), reduced kidney size7, 8), and impaired renal function9, 10), highlighting the long-term consequences of preterm birth on renal health. The cause of impaired renal development after preterm birth and the mechanisms through which it may predispose adults to renal disease are still largely unknown; exposure to oxygen in the extrauterine environment may be a contributing factor11-13). At birth, infants are exposed to oxygen concentrations far exceeding the intrauterine levels14), causing oxidative stress15, 16), which they are particularly susceptible to because they have low antioxidant levels17, 18). Macrophage infiltration, reactive oxygen species (ROS) activity, and renin-angiotensin system activation are essential in renal injury pathogenesis19). Oxidative stress is a common pathway leading to chronic renal damage, with damage to cells, tissues, and organs caused by ROS20). It has been implicated in numerous commonly occurring preterm birth-related conditions, including retinopathy of prematurity (ROP)21). High oxygen load increases oxidative stress in rats, resulting in glomerular developmental disorders22).Alternatively, hydrogen reduces oxidative stress, effectively protecting the tissue from injury. Hydrogen selectively decreases the production of hydroxyl radical and peroxynitrite, which are two of the most cytotoxic ROS, and protects against oxidative stress23). In rats, hydrogen-rich water (HW) and hydrogen saline significantly attenuate renal ischemia-reperfusion injury and reduce the serum levels of 8-OHdG (a biomarker of oxidative DNA damage)24, 25). Several nephrology studies have also demonstrated the efficacy of hydrogen.In this study, we aimed to investigate the effect of hydrogen on oxidative stress and glomerular developmental disorders in newborn rats with a high-concentration oxygen load.Materials and MethodsAnimalsThe Juntendo University Animal Care Facility (Tokyo, Japan) approved all of our study proce-dures. Female Sprague-Dawley rats at gestational day 19 were purchased from Nihon SLC, Co., Ltd.