Excessive Mitochondrial Copper Load Driven by Upregulated Copper Transporter 1 Contributes to Mitochondrial Dysfunction and Renal Fibrosis

Background: Copper is an essential trace element for eukaryotes. Our previous study indicated that intracellular copper overload plays an important role in renal fibrosis. However, the underlying mechanism remains largely unknown. In this study, we found copper ions in cells were mainly accumulated in mitochondria, which damage the structure and function of mitochondria. Furthermore, copper transporter 1(CTR1), the major transporter for copper influx, was significantly increased in fibrotic kidneys. The activity of cytochrome c oxidase (COX), a copper coenzyme mediating the final step in the electron transport chain, was decreased. Downregulation of CTR1 decreased mitochondrial copper overload. Therefore, we proposed that CTR1 might be involved in mitochondrial copper overload and renal fibrosis. Methods: Expression of CTR1 was examined in fibrotic kidneys of patients, ischemia-reperfusion injury (IRI 28d) mice model and TGF-β1-treated tubular epithelial cells. We also use stable CTR1 knockdown cell lines in vitro, and in vivo, we use CTR1 transgenic mice subjected to IRI operation. Copper chelator tetrathiomolybdate (TM), ICP-MS, mitoSOX Red, electron microscopy, realtime-PCR and western blot analysis were applied in the current study. Results: We found that stimulated by TGF-β1, COX activity was declined. Mitochondria and cytosol, especially mitochondria, accumulated a large amount of copper in fibrotic kidney tissues. Furthermore, we found CTR1 expression was increased in fibrotic kidneys from chronic kidney disease patients, in vivo and in vitro. More importantly, compared to WT mice, CTR1+/- mice subjected to ischemia-reperfusion injury (IRI) had reduced mitochondrial copper level, and ameliorated mitochondrial function and renal fibrosis, as evidenced by improving mitochondrial structure, inhibiting mtROS production and reducing expression of α-smooth muscle actin (α-SMA), collagen I and fibronectin. In addition, mitochondrial function and kidney fibrosis were improved in IRI mice kidney after TM treatment. Conclusions: Collectively, our study showed that copper overload in mitochondria could damage mitochondrial function and lead to renal fibrosis. Among which CTR1 plays an incomparable role in the mitochondrial copper overload. Copper overload inhibits the activity of COX and impairs mitochondria, subsequently leading to renal fibrosis.