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.
Highlights
Synthesis and secretion of the systemic iron-regulatory hormone hepcidin by the liver is regulated by iron stores within macrophages, inflammation, hypoxia, and erythropoiesis
Several studies indicate that hepcidin protects against certain forms of kidney injury caused by oxidative stress (Scindia et al 2015; van Swelm et al 2016, 2018), and possibly interferes with cell death signaling or chelates Fe2+ (Farnaud et al 2006, 2008; Gerardi et al 2005), the function of hepcidin locally expressed in the distal nephron is not understood
We demonstrate distinct effects of hepcidin on Fe2+- and Cd2+-induced oxidative stress and death in mIMCD3 and mCCD(cl.1) cells (Fig. 7)
Summary
Synthesis and secretion of the systemic iron-regulatory hormone hepcidin (rodent gene name Hamp1) by the liver is regulated by iron stores within macrophages, inflammation, hypoxia, and erythropoiesis (reviewed in Ganz and Nemeth 2012). Binding of circulating hepcidin to its target protein ferroportin-1 on enterocytes and macrophages leads to internalization and degradation of the hepcidin-ferroportin-1 complex by ubiquitination, thereby inhibiting the efflux of iron from intestinal enterocytes into the blood as well as preventing the release of stored iron from macrophages (Nemeth et al 2004). Systemic iron overload induced by genetic diseases, such as hereditary haemochromatosis or β-thalassemia major result in renal iron deposition and kidney injury involving oxidative stress induced by the Fenton metal ion Fe2+ (Halliwell and Gutteridge 2015), various forms of cell death or inflammation (reviewed in van Swelm et al 2020). The distal nephron expresses a variety of Cd2+ entry pathways, including iron response element (IRE)-regulated F e2+ transporters (reviewed in Smith and Thévenod 2009; Thévenod and Wolff 2016). We assessed roles of hepcidin in Fe2+- and Cd2+-induced ROS formation and cell injury in the mouse kidney cortical and inner medullary collecting duct (CD) cell lines mCCD(cl.1) and mIMCD3
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