Abstract: BACKGROUND: Hexafluoropropylene oxide dimer acid (also known as GenX) is widely used as a novel substitute for perfluorooctanoic acid in coatings, inks, antioxidants, and other fields. However, GenX exhibits multi-organ toxicity, and exposure to GenX can lead to elevated blood urea nitrogen levels and renal pathological damage in rats. 
OBJECTIVE: To investigate the toxic effects and mechanism of low-dose GenX exposure during pregnancy on the kidney of offspring mice.
METHODS: (1) Animal experiment: Sexually mature 8-week-old female BALB/c mice were randomly divided into normal control group and GenX group after mating with male mice and showing vaginal plug, with six mice in each group. The control group was gavaged with ddH2O, while the GenX group was gavaged with GenX (2 mg/kg/d) until birth. Kidney tissues of male and female offspring mice were collected at 3 and 8 weeks after birth. Hematoxylin-eosin staining and Masson staining were used to explore the effects of GenX on the pathological structure and fibrosis of the kidney in offspring mice of different sexes. Immunohistochemical staining was used to detect the expression of inflammatory factors (tumor necrosis factor α and interleukin 6) in renal tissues. β-galactosidase staining was used to detect renal aging. Immunofluorescence staining of calcineurin E and vimentin expression was used to detect renal epithelial-mesenchymal transition, and immunofluorescence staining was used to detect transforming growth factor β1 expression. (2) Cell experiment: Renal tubular epithelial cells HK-2 were divided into three groups: control group with no treatment, GenX group treated with 600 μmol/L GenX for 72 hours, and GenX+rapamycin group with 10 nmol/L rapamycin pretreatment for 1 hour followed by addition of 600 μmol/L GenX for 72 hours. Cell supernatant was taken for use in the following experiment. Kidney fibroblasts NRK-49F were treated in three groups, and the culture supernatants of the above three groups were added and treated for 48 hours. Immunofluorescence staining was performed to detect the expression of fibroblast activation markers α-smooth muscle actin and fibronectin.
RESULTS AND CONCLUSION: (1) Animal experiment: Exposure to low-dose GenX during pregnancy could cause structural damage and tissue fibrosis in the kidneys of female and male offspring mice, trigger inflammatory responses and renal senescence in female and male offspring mice, induce the development of epithelial-mesenchymal transition in the kidneys of female and male offspring mice, and increase the expression of transforming growth factor β1 in the kidneys of female and male offspring mice. (2) Cell experiment: Immunofluorescence staining showed that hexafluoropropylene oxide dimer acid-treated HK-2 cell culture supernatants significantly increased α-smooth muscle actin and fibronectin expression in NRK-49F cells, but this effect was inhibited by the senescence inhibitor rapamycin. To conclude, GenX exposure during pregnancy is toxic to the kidneys of offspring mice, causing the development of renal inflammation and fibrosis, mainly through accelerating the senescence of renal tubular epithelial cells, promoting epithelial-mesenchymal transition, and promoting the proliferation and activation of fibroblasts, and ultimately leading to the development of renal fibrosis.

Key words: GenX, renal fibrosis, aging, epithelial-mesenchymal transition, renal toxicity, offspring mice