Maternal exposure to di-n-butyl phthalate (DBP) induces renal fibrosis in adult rat offspring

Abstract

This study was to determine the impact of maternal exposure to di-n-butyl phthalate (DBP) on renal development and fibrosis in adult offspring. Pregnant rats received DBP at a dose of 850 mg/kg BW/day by oral perfusion during gestational days 14-18. In DBP exposed newborn offspring, gross observation and histopathological examination revealed the dysplasia of kidney. The expression of genes related to renal development was also changed. In DBP exposed adult offspring, histopathological examination and Masson's trichrome staining revealed the pathological changes of renal fibrosis. Furthermore, higher expression levels of transforming growth factor- β (TGF-β) and alpha-smooth muscle actin (α-SMA) were also detected. In vitro studies reveal that DBP promoted the activation of NRK49F cells and G2/M arrest in NRK52E cells at a sublethal dose. The effect of DBP on these cell lines was linked to the generation of oxidative stress. In addition, DBP induced oxidative stress in both renal fibroblasts and tubular epithelial cells, whereas vitamin C ameliorated the changes caused by DBP. In conclusion, our results showed that prenatal exposure to DBP may generate oxidative stress in both renal fibroblasts and tubular epithelial cells, leading to kidney dysplasia and renal fibrosis.

Keywords: di-n-butyl phthalate (DBP); environmental endocrine-disrupting compounds (EEDs); kidney dysplasia; oxidative stress; renal fibrosis.

Figure 1. Maternal exposure to DBP induces kidney dysplasia in rat offspring on PND1

(A) The body weight of the rat offspring in the DBP-exposed group and DBP-unexposed controls on PND1. (B) Gross images of the kidneys in the DBP-exposed group and DBP-unexposed controls on PND1. (C) The organ/body weight ratio of the kidneys in the DBP-exposed group and the DBP-unexposed control group on PND1. (D) H&E stained images of kidney tissue showed differences between the control and DBP-exposed offspring on PND1. (E) Real-time PCR assessed the expression of genes involved in renal development (Foxd1, Gdnf, Pax2, Wnt11, Bmp4, Cdh11, Calm1, Cfl1, Ywhab) in the kidneys of DBP-exposed rats and DBP-unexposed controls on PND1. Data represent mean ± SD. n = 6 rats per group, *P < 0.05.

Figure 2. Maternal exposure to DBP leads to renal fibrosis in adult offspring

(A) H&E and Masson trichrome stained images of kidneys in the DBP-exposed adult offspring and DBP-unexposed controls. (B) Represented immunohistochemical localization of α-SMA, fibronectin and TGF-β in the kidneys of DBP-exposed adult offspring and unexposed controls. (C) The average optical density of α-SMA, fibronectin and TGF-β in the kidneys of DBP-exposed adult offspring and unexposed controls. (D) Western blot showing different α-SMA, fibronectin and TGF-β protein levels of kidneys in DBP-exposed adult offspring and unexposed controls. (E) Densitometric quantification of α-SMA, fibronectin and TGF-β proteins in the kidneys of DBP-exposed adult offspring and unexposed controls. (F) Real-time PCR assessed the expression of α-SMA (Acta), fibronectin (Fn) and TGF-β (Tgfb) in the kidneys of DBP-exposed adult offspring and unexposed controls. Data represent mean ± SD. n = 6 rats per group, *P < 0.05.

Figure 3. DBP promotes the proliferation and activation of NRK49F cells at a sublethal dose

(A) CCK-8 cell proliferation assays showing different proliferation between NRK49F cells exposed to various concentrations of DBP (1, 10, 100 μmol/L). (B) Hoechst 33342 dye stained images of NRK49F cells exposed to various concentrations of DBP (1, 10, 100 μmol/L). (C) Western blot showing different α-SMA, fibronectin and TGF-β protein levels of NRK49F cells in DBP-exposed, vitamin C-exposed group and unexposed controls. (D) Densitometric quantification of α-SMA, fibronectin and TGF-β proteins levels of NRK49F cells in DBP-exposed, vitamin C-exposed group and unexposed controls. (E) Real-time PCR assessed the expression of α-SMA (Acta), fibronectin (Fn) and TGF-β (Tgfb) of NRK49F cells in DBP-exposed, vitamin C-exposed group and unexposed controls. Data represent mean ± SD. n = 6 rats per group, *P < 0.05.

Figure 4. DBP inhibits NRK52E cell growth and induces G2/M arrest and the overproduction of TGF-β in NRK52E cells at a sublethal dose

(A) CCK-8 cell proliferation assays showing different proliferation between NRK52E cells exposed to various concentrations of DBP (1, 10, 100 μmol/L). (B) Dual immunofluorescence of Ki67 (red) and p-H3 (green) in NRK52E cells of DBP-exposed, vitamin C-exposed group and unexposed controls. DAPI (blue) was used for nuclear staining. (C) Hoechst 33342 dye stained images of NRK52E cells exposed to various concentrations of DBP (1, 10, 100 μmol/L). (D) Western blot showing different TGF-β and p21 protein levels of NRK52E cells in DBP-exposed, vitamin C-exposed group and unexposed controls. (E) Densitometric quantification of TGF-β and p21 protein levels of NRK52E cells in DBP-exposed, vitamin C-exposed group and unexposed controls. (F) Real-time PCR assessed the expression of TGF-β (Tgfb) and p21(Cdkn1a) of NRK52E cells in DBP-exposed, vitamin C-exposed group and unexposed controls. Data represent mean ± SD. n = 6 rats per group, *P < 0.05.

Figure 5. DBP generates oxidative stress and reduces the expression of the gene responsible for the prevention of oxidative activity in NRK49F and NRK52E cells

(A) Measurement of intracellular ROS in NRK49F cells by DCFH-DA dye showed the quantity of ROS increased significantly after treatment with various concentrations of DBP (1, 10, 100 μmol/L). (B) Measurement of intracellular ROS in NRK52E cells by DCFH-DA dye showed the quantity of ROS increased significantly after treatment with various concentrations of DBP (1, 10, 100 μmol/L). (C) Real-time PCR assessed the expression of antioxidant genes (Gpx1, Cat, GR, Gst) of NRK49F cells exposed to various concentrations of DBP (1, 10, 100 μmol/L). (D) Real-time PCR assessed the expression of antioxidant genes (Gpx1, Cat, GR, Gst) of NRK52E cells exposed to various concentrations of DBP (1, 10, 100 μmol/L). (E) Measurement of intracellular ROS by DCFH-DA dye in NRK49F cells in DBP-exposed, vitamin C-exposed group and unexposed controls. (F) Measurement of intracellular ROS by DCFH-DA dye in NRK52E cells in DBP-exposed, vitamin C-exposed group and unexposed controls. (G) Real-time PCR assessed the expression of antioxidant genes (Gpx1, Cat, GR, Gst) of NRK49F cells in DBP-exposed, vitamin C-exposed group and unexposed controls. (H) Real-time PCR assessed the expression of antioxidant genes (Gpx1, Cat, GR, Gst) of NRK52E cells in DBP-exposed, vitamin C-exposed group and unexposed controls. Data represent mean ± SD. n = 6 rats per group, *P < 0.05.