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C-FGF23 peptide alleviates hypoferremia
Table 1. Effects of lipopolysaccharide (LPS) and fibroblast growth factor 23 (FGF23) blocking peptide on hematologic parameters in mice.
WBC, x109/L
Lymphocyte, x109/L Neutrophil, x109/L RBC, x1012/L
Hb, g/dL Hematocrit, % RDW, %
MCV, fL
Ctl
2.47±0.41
1.73±0.66 0.19±0.22 9.80±0.35 14.18±0.41 45.24±1.55 18.14±1.18 46.00±0.00
LPS
2.79±0.78
1.22±0.30 1.49±0.53a*** 10.85±0.59a** 15.23±0.94a* 48.96±2.49a** 17.41±0.43 45.60±1.65
FGF23 BL
2.16±0.80
2.09±0.53 0.23±0.22
FGF23 BL + LPS
1.72±0.76b**
0.80±0.36b*, c*** 0.81±0.45b**, c*
10.09±0.22 10.28±0.68b* 14.62±0.69 14.45±0.67b* 46.96±1.12 46.32±2.23b* 17.50±0.37 16.73±0.50b**, c* 46.40±0.55 45.20±1.48
aValues of LPS or FGF23 BL (FGF23 blocking peptide) differ significantly compared to control (vehicle) (Ctl). bValues of FGF23 BL+LPS differ significantly compared to LPS. cValues of FGF23 BL+LPS differ significantly compared to FGF23 BL. *P<0.05, **P<0.01, ***P<0.001.
regulation of phosphate homeostasis by promoting phosphate excretion.
Acute inflammation induces hypoferremia and downregulates Epo expression
Inflammation is a major stimulus for hepcidin synthe- sis in the liver. Specifically, treatment of hepatocytes with LPS leads to increase in hepcidin levels.32,33 In agree- ment with published studies, our data show that hep- cidin expression started rising 1 h after LPS treatment with significant upregulation between 4-6 h after LPS (Figure 3A), consistent with the presence of inflamma- tion (Figure 1A-C), and returned to basal levels after res- olution of inflammation (Figure 3A). The increase in hep- cidin resulted in progressive and significant reduction in circulating iron and transferrin saturation levels (Figure 3B and C), followed by recovery from hypoferremia once inflammation was resolved and hepcidin levels returned to normal. This drop in serum iron levels was accompanied by a significant decrease in ferroportin expression in liver and spleen (Figure 3D and E), leading to iron retention in these organs (Figure 3F and Online Supplementary Figure S2) and increased spleen weight (Figure 3G). In addition, expression of the acute phase protein lipocalin 2 (Lcn2), which plays a role as an iron sequester,34 was increased after 4 h of LPS and remained elevated up to 24 h post LPS treatment (Figure 3H). These data demonstrate that LPS induces hypoferremia associated with downregulation of ferroportin and iron retention in liver and spleen.
Studies have shown that pro-inflammatory cytokines, such as IL-6, TNF-α, and IL-1β, increased by inflamma- tion and/or infection, inhibit renal erythropoietin (Epo) production, the primary regulator of erythropoiesis, and therefore suppress erythropoiesis.35-37 This is known as anemia of inflammation. Specifically, it has been shown that administration of LPS in rats significantly sup- pressed renal Epo gene expression.36 In our studies, we found that renal Epo expression was significantly down- regulated after 4-6 h (Figure 3I).
Inhibition of FGF23 signaling alleviates hypoferremia induced by acute inflammation
It has been reported that the C-terminal tail of FGF23 mediates the binding of FGF23 to the FGFR-Klotho com- plex.27,38 Studies have shown that the 72-aa-long C-termi- nal tail of FGF23 (residues Ser180-Ile251), which corre- sponds to the C-terminal fragment of FGF23 generated by proteolytic cleavage at the RXXR motif, impairs FGF23
signaling and activity by acting as endogenous inhibitor of the full-length FGF23 for binding to the FGFR-Klotho complex.27 We previously reported that inhibition of FGF23 signaling using this FGF23 blocking peptide (BL) ameliorates iron deficiency in a mouse model of CKD.29 Since FGF23 significantly increases in response to LPS, here we assessed the effect of inhibiting FGF23 signaling in a mouse model of hypoferremia induced by acute inflammation. Based on the results of our longitudinal studies, we opted to assess the effect of FGF23 signaling inhibition 4 h post LPS treatment. At this time point, we observed maximum induction of FGF23 and hepcidin, associated with a significant decrease in serum iron, transferrin saturation, and erythropoietin, during upregu- lation of pro-inflammatory cytokines.
Our data show that the increase in FGF23 in response to LPS was significantly reduced after inhibition of FGF23 signaling (Figure 4A-C). Administration of LPS triggered an acute inflammatory response (Figure 5A-C) that led to induction of hepcidin (Figure 5D and E). However, blocking FGF23 signaling significantly decreased hepatic expression of TNF-α without affecting IL-6 and IL-1β mRNA expression in LPS-treated mice (Figure 5A-C). Importantly, the increase in hepatic and circulating hepcidin levels induced by LPS was signifi- cantly reduced when FGF23 signaling was impaired (Figure 5D and E). We also found that inhibition of FGF23 signaling did not impact phosphorylation of STAT3, the main intracellular regulator of hepcidin induction by IL6, as treatment with cFGF23 did not affect LPS-induced upregulation of hepatic STAT3 phos- phorylation (Online Supplementary Figure S3). Therefore, suppression of hepcidin by cFGF23 appears to be inde- pendent of the IL6/STAT3 pathway. These data suggest that inhibiting FGF23 signaling reduces LPS-induced hep- cidin in mice with normal kidney function.
Increased hepcidin leads to iron sequestration in tis- sues and subsequent decrease in circulating iron lev- els.7,8,39 As expected, LPS elicited hypoferremia after 4 h with a significant decrease in serum iron and transferrin saturation (Figure 6A and B), associated with a robust reduction in liver and spleen ferroportin (Fpn) mRNA (Figure 6C and D). Downregulation of ferroportin result- ed in increased hepatic and splenic iron content (Figure 6E and F), which correlated with increased hepatic expression of the iron storage protein ferritin H (Fth) (Figure 6G) and the iron sequester lipocalin 2 (Lcn2) (Figure 6H). The number of circulating neutrophils, which are the main source of lipocalin secretion, was
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