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R. Agoro et al.
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Figure 3. Effect of lipopolysaccharide (LPS) on iron homeostasis and renal Epo mRNA expression. C57BL/6J mice were injected intraperitoneally (i.p.) with saline (0.9% NaCl, indicated as Vehicle) or LPS (50 mg/kg). Samples were collected at 0, 1, 2, 4, 6, 12, and 24 hours (h) after treatment. (A) Quantitative real-time poly- merase chain reaction (qRT-PCR) for hepcidin expression in liver. (B) Serum iron and (C) serum transferrin saturation. (D and E) qRT-PCR for ferroportin (Fpn) expres- sion in (D) liver and (E) spleen. (F) Iron content in spleen. (G) Spleen weight normalized to the body weight of the animals. (H) qRT-PCR for lipocalin (Lcn2) in liver. (I) qRT-PCR for renal Epo mRNA expression. Data are expressed as fold change (2-DDCt) relative to housekeeping genes Gapdh or Hprt. Samples were measured in dupli- cates (vehicle, n=3-4; LPS, n=5-8). Data are represented as mean±standard deviation. All data were analyzed for normality with Shapiro-Wilk test and equivalence of variance using Levene’s test. The samples not in normal distribution were analyzed with non-parametric Kruskal-Wallis test (A and B, D-I). When the samples showed normal distribution, two-way ANOVA was performed with Bonferroni’s multiple comparison test in each vehicle- or LPS-treated group compared to 0 h (C). ns: not significant, *P<0.05, **P<0.01, ***P<0.001 compared to 0 h.
also increased in response to LPS (Table 1). Importantly, inhibition of FGF23 signaling increased serum iron and transferrin saturation levels in both basal and LPS condi- tions (Figure 6A and B), and prevented LPS-induced liver and spleen iron sequestration (Figure 6E and F). Circulating neutrophils and expression of ferritin H and lipocalin 2 in the liver were also significantly reduced after administration of the FGF23 C-tail blocking peptide in LPS-treated mice (Table 1 and Figure 6G and H). However, blocking FGF23 signaling did not change the expression of liver and spleen ferroportin in response to LPS (Figure 6C and D), but in basal conditions it selec- tively induced splenic ferroportin mRNA and protein expression compared to controls (Figure 6D and Online Supplementary Figure S4). Taken together, these data sug- gest that inhibition of FGF23 signaling can alleviate
hypoferremia induced by acute inflammation in mice in the absence of renal disease.
Inhibition of FGF23 signaling increases renal and EPO andEpoR
Consistent with previously published data,36 our studies show that LPS significantly downregulated renal Epo and EpoR expression after 4 h (Figure 7A and B). We also found that renal Hif2α mRNA was significantly decreased after LPS treatment (Figure 7C), in line with the role of HIF2 as a regulator of Epo synthesis and iron metabolism.40 Importantly, inhibition of FGF23 signaling prevented the downregulation of renal Epo, EpoR, and Hif2α expression caused by LPS (Figure 7A-C). We previously reported that treatment with the FGF23 C-tail blocking peptide signifi- cantly increases circulating Epo levels in mice.29 Here, we
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haematologica | 2021; 106(2)