Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium

Abstract

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5–5 μmol/l) and/or Fe2+ (50–100 μmol/l) for 4–24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

An Overlooked Hepcidin-cadmium Connection

“…Biological experiments indicate that unlike the MTs, hepcidin apparently does not provide a cytoprotective effect. As opposed to MTs, hepcidin silencing decreases Cd(II) toxicity while overexpression of hepcidin decreases cell viability in the presence of cadmium [16].…”

“…What is interesting, the hepcidin gene (Hamp1) silencing decreases the Cd 2+ toxicity in mIMCD3 cells. Similarly, overexpression of Hamp1 increases the Cd 2+ toxicity [16]. A full mechanism for that is yet unknown.…”

An Overlooked Hepcidin–Cadmium Connection

“…Biological experiments indicate that unlike the MTs, hepcidin apparently does not provide a cytoprotective effect. As opposed to MTs, hepcidin silencing decreases Cd(II) toxicity while overexpression of hepcidin decreases cell viability in the presence of cadmium [ 23 ]. Interaction of hepcidin and cadmium inside the cell (whether direct or indirect) results in the generation of reactive oxygen species.…”

“…Interestingly, hepcidin-gene ( Hamp1 ) silencing decreases Cd 2+ toxicity in mice inner medullary collecting duct (mIMCD 3 ) cells. Similarly, overexpression of Hamp1 increases Cd 2+ toxicity [ 23 ]. A full mechanism for this phenomenon is still unknown.…”

Cadmium transport by mammalian ATP-binding cassette transporters

“…Finally, genetic manipulation of ABCB6 revealed positive regulation of expression and activity of catalase, a heme-containing enzyme that catalyzes the decomposition of hydrogen peroxide (Baker et al 2023 ), suggesting increased availability of heme—as a direct result of ABCB6-mediated porphyrin transport—stabilizes catalase and thus provides augmented protection against oxidative stress (Lynch et al 2009 ). This mechanism could be implicated in Cd toxicity, in which oxidative stress partly through inhibition of catalase is elicited (Lee et al 2024 ; Probst et al 2021 ).…”