Supplementary Material for: Green tea polyphenols alleviate kidney injury induced by di(2-ethylhexyl) phthalate in mice

Background: Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer. Studies have revealed that DEHP exposure can cause kidney damage. Green tea is among the most popular beverages in China. Green tea polyphenols (GTPs) have been proven to have therapeutic effects on organ damage induced by heavy metal exposure. However, few studies have reported on GTP relieving DEHP-induced kidney damage. Methods: C57BL/6J male mice aged 6–8 weeks were treated with distilled water (control group), 1500 mg/kg/d DEHP + corn oil (model group), 1500 mg/kg/d DEHP + corn oil + 70 mg/kg GTP (treatment group), corn oil (oil group), and 70 mg/kg GTP (GTP group) by gavage for 8 weeks, respectively. The renal function of mice and renal tissue histopathology of each group were evaluated. The renal tissues of mice in the model, treatment, and control groups were analyzed using high-throughput sequencing. We calculated the differentially expressed miRNAs and mRNAs using the limma R package, the CIBERSORT algorithm was used to predict immune infiltration, the starBase database was used to screen the miRNA–mRNA regulatory axis, and immunohistochemical analyses were performed to verify protein expression. Results: GTP alleviated the deterioration of renal function, renal inflammation and fibrosis, and mitochondrial and endoplasmic reticulum lesions induced by DEHP in mice. Differential immune infiltrations of plasma, dendritic, T, and B cells were noted between the model and treatment groups. We found that three differentially expressed miRNAs (mmu-miR-383-5p, mmu-miR-152-3p, and mmu-miR-144-3p), three differentially expressed mRNAs (Ddit4, Dusp1, and Snx18), and three differentially expressed proteins (Ddit4, Dusp1, and Snx18) played crucial roles in the miRNA–mRNA–protein regulatory axes when GTPs mitigate DEHP-induced kidney damage in mice. Conclusion: GTP can alleviate DEHP-induced kidney damage and regulate immune cell infiltration. We screened four important miRNA–mRNA–protein regulatory axes of GTP mitigating DEHP-induced kidney damage in mice.