Proximal tubule H-ferritin mediates iron trafficking in acute kidney injury
Abolfazl Zarjou, … , Lukas C. Kuhn, Anupam Agarwal
Abolfazl Zarjou, … , Lukas C. Kuhn, Anupam Agarwal
Published September 9, 2013
Citation Information: J Clin Invest. 2013;123(10):4423-4434. https://doi.org/10.1172/JCI67867.
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Research Article Nephrology

Proximal tubule H-ferritin mediates iron trafficking in acute kidney injury

Abstract

Ferritin plays a central role in iron metabolism and is made of 24 subunits of 2 types: heavy chain and light chain. The ferritin heavy chain (FtH) has ferroxidase activity that is required for iron incorporation and limiting toxicity. The purpose of this study was to investigate the role of FtH in acute kidney injury (AKI) and renal iron handling by using proximal tubule–specific FtH-knockout mice (FtHPT–/– mice). FtHPT–/– mice had significant mortality, worse structural and functional renal injury, and increased levels of apoptosis in rhabdomyolysis and cisplatin-induced AKI, despite significantly higher expression of heme oxygenase-1, an antioxidant and cytoprotective enzyme. While expression of divalent metal transporter-1 was unaffected, expression of ferroportin (FPN) was significantly lower under both basal and rhabdomyolysis-induced AKI in FtHPT–/– mice. Apical localization of FPN was disrupted after AKI to a diffuse cytosolic and basolateral pattern. FtH, regardless of iron content and ferroxidase activity, induced FPN. Interestingly, urinary levels of the iron acceptor proteins neutrophil gelatinase–associated lipocalin, hemopexin, and transferrin were increased in FtHPT–/– mice after AKI. These results underscore the protective role of FtH and reveal the critical role of proximal tubule FtH in iron trafficking in AKI.

Authors

Abolfazl Zarjou, Subhashini Bolisetty, Reny Joseph, Amie Traylor, Eugene O. Apostolov, Paolo Arosio, Jozsef Balla, Jill Verlander, Deepak Darshan, Lukas C. Kuhn, Anupam Agarwal

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Figure 6

FtH regulates FPN expression in renal proximal tubules.

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FtH regulates FPN expression in renal proximal tubules. (A) Whole kidney...
(A) Whole kidney lysates from HO-1+/+ and HO-1–/– mice were analyzed for FPN, HO-1, and FtH levels by Western blot. Membranes were reprobed for GAPDH to demonstrate equal loading. (B) Kidney FPN and FtH expression was analyzed by densitometry analysis, normalized to GAPDH, and expressed as mean ± SEM. *P < 0.05 vs HO-1+/+. (C) FtHPT+/+ proximal tubular epithelial cells were treated in vitro with apoferritin for 8 hours and analyzed for FPN expression by real-time PCR. Data are expressed as fold change relative to vehicle-treated cells. *P < 0.05. (D) Proximal tubular epithelial cells were treated with the indicated doses of apoferritin for 16 hours and analyzed for FPN and FtH expression by Western blot. GAPDH served as a loading control. (E) Proximal tubular epithelial cells were treated with vehicle, FtH, or FtH-M for 16 hours and analyzed for FPN and FtH expression by Western blot. GAPDH served as a loading control. Results are representative of 3 independent experiments. (F) Immunocytochemistry for ZO-1 expression on cells grown on Transwell filters, confirming polarization. Original magnification, ×180. (G) Proximal tubular epithelial cells were treated with hepcidin for the indicated times and analyzed for FPN expression by Western blot. (H) Proximal tubular cells grown on Transwell filters were pretreated with vehicle (control) or hepcidin for 48 hours, 55Fe was administered to the apical compartment for 2 hours, and cellular 55Fe levels were measured and expressed as picomoles of iron per milligram protein. *P < 0.05 vs. control.