Hepcidin-2 in mouse urine as a candidate radiation-responsive molecule

Abstract

We used high-performance liquid chromatography to separate urine obtained from whole-body gamma-irradiated mice (4 Gy) before analyzing each fraction with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to identify radiation-responsive molecules. We identified two candidates: hepcidin antimicrobial peptide 2 (hepcidin-2) and peptide fragments of kidney androgen-regulated protein (KAP). We observed that peak increases of hepcidin-2 in urine were delayed in a dose-dependent manner (1 Gy and above); however, the amount of KAP peptide fragments showed no correlation with radiation dose. In addition, an increase in hepcidin-2 after exposure to relatively low radiation doses (0.25 and 0.5 Gy, respectively) was biphasic (at 8-48 h and 120-168 h, respectively, after irradiation). The increase in hepcidin-2 paralleled an increase in hepcidin-2 gene (Hamp2) mRNA levels in the liver. These results suggest that radiation exposure directly or indirectly induces urinary excretion of hepcidin-2 at least in part by the upregulation of Hamp2 mRNA in the liver.

Keywords: hepcidin-2; mass spectrometry; radiation-responsive molecules; urine.

Fig. 1.

( A ) Representative total ion intensity in each fraction of mouse urine individually separated by high-performance liquid chromatography (HPLC): bold line, sham-irradiated; broken line, 24 h after 4 Gy irradiation. The sum intensity of each fraction is shown by retention time. Arrows show the changes in response to radiation. ( B , C and D ) Representative matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry spectra of HPLC fractions no. 24, 35 and 46, respectively. The arrows represent the peak of angiotensin II ( m/z 1046) as the internal control, and the arrowheads represent m/z 1250 (B), 1721 (C) and 2821 (D), respectively. ( E ) Representative tandem mass spectrometry (MS/MS) spectra of m/z 1721 and (F) 2821. ( G ) Full MS/MS spectra of m/z 2821 were obtained from samples treated with dithiothreitol.

Fig. 2.

( A ) Representative matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry spectra from unfractionated urine, which we concentrated and deionized with OMIX tips. We collected urine samples from mice before irradiation (bottom) or 24 h (middle) or 48 h (top) after 4 Gy irradiation. Arrow: peptide fragment of uromodulin ( m/z 2001); arrowhead: hepcidin-2 ( m/z 2821). ( B ) Representative tandem spectrometry spectrum of m/z 2001.

Fig. 3.

( A ) Urinary excretion of hepcidin-2 by high-dose irradiation. ( B ) Urinary excretion of hepcidin-2 by low-dose irradiation. We used the uromodulin peptide fragment as normalization, and we calculated relative intensity compared with the levels found in mice prior to sham irradiation. * P < 0.05, ** P < 0.01, compared with sham-irradiated mice at the same time-point.

Fig. 4.

Expression of Hamp and Hamp2 genes in the liver after whole-body irradiation. We analyzed Hamp2 ( A ) and Hamp ( B ) expression levels with quantitative reverse transcription-polymerase chain reaction. We calculated the relative expression levels compared with the levels found in sham-irradiated mice. * P < 0.05, ** P < 0.01.