Combined effects of nanoplastics and 3-BHA at environmentally relevant concentrations significantly aggravated kidney injury via TGF-β/SMAD signaling pathway in mice

The increasing presence of nanoplastics (NPs) and synthetic antioxidants like 3-tert -Butyl-4-hydroxyanisole (3-BHA) in the environment has attracted widespread attention about their combined toxicological effects on human health, particularly on renal function. This study explored to the combined impacts of NPs and 3-BHA at environmentally relevant concentrations on sub-chronic kidney injury in mice. Firstly, our results confirmed that the accumulation of 80 nm NPs in renal tissues, leading to structural abnormalities such as reduced mitochondrial cristae and increased empty bubbles in mice by transmission electron microscope (TEM) analysis. Secondly, NPs and 3-BHA co-treatment resulted in significant renal developmental abnormalities including decreased kidney weight, as well as reduced organ index of kidney/body weight in mice. Besides, the histopathological staining revealed that severe renal damage characterized by thickened tubular epithelium, glomerular atrophy, and inflammatory cell infiltration were observed in kidney tissues after co-exposure. Additionally, co-exposure increased collagen fiber deposition and enhanced renal fibrosis, which showing elevated expression levels of renal function markers such as BUN, CRE, Cys-C and β2-MG. Oxidative stress and renal apoptosis were significantly aggravated, as evidenced by increased CAT, GSH-Px, and SOD levels together with TUNEL staining and expressions of apoptosis-related genes. Furthermore, co-exposure of NPs and 3-BHA caused the activation of TGF-β/Smad signaling pathway, promoting epithelial-to-mesenchymal transition (EMT) and extracellular matrix deposition, which are key processes in renal fibrosis. Thirdly, RNA-Seq analysis identified disruptions in energy metabolism and redox balance, with terms associated with oxidative phosphorylation and mitochondrial ATP synthesis were significantly enriched after NPs and 3-BHA co-exposure. Finally, pharmacological inhibition of TGF-β/Smad signaling using hydrochlorothiazide (HCTZ) could partially rescue the renal damage, cell apoptosis, inflammation and fibrosis after co-treatment. In summary, these results demonstrated the synergistic nephrotoxic effects of nanoplastics and 3-BHA in mice, which mainly mediated through oxidative stress, inflammation/apoptosis and TGF-β/SMAD signaling pathway. These information point to the critical need for further exploration of the combined effects of environmental pollutants on human kidney function.