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C-FGF23 peptide alleviates hypoferremia
respectively.58,62 The lack of hepatic FGFR4 signaling inves- tigation is a limitation of our study. Therefore, further experiments are required to delineate the hepatic FGFR signaling that contributes to hypoferremia. Mice deficient in Fgf23 (Fgf23-/-) exhibit aberrant bone mineralization along with high serum phosphate, calcium, and vitamin D levels, tissue and vascular calcifications, early lethality within 12 weeks after birth,19 in addition to inflammation and dysregulation of iron homeostasis (data not shown), thus limiting the possibility of using these mice to investi- gate the impact of FGF23 in response to hypoferremia. Here we evaluated the effect of pharmacologic inhibition of FGF23 signaling using the C-tail FGF23 in a mouse model of LPS-induced hypoferremia. Binding and cell sig- naling studies have shown that the C-tail of FGF23 can inhibit FGF23 signaling and antagonize its phosphaturic activity in vivo by competing with the intact FGF23 for binding to the FGFR-Klotho complex.27 Moreover, we have previously successfully used the C-tail FGF23 in a CKD mouse model to rescue renal anemia and iron defi- ciency and attenuate chronic inflammation.29
CKD patients, as well as mice with 5/6 nephrectomy, exhibit elevation in FGF23 levels associated with down- regulation of renal Klotho expression. Our longitudinal studies showed that serum phosphate levels significantly increased the first 2 h of LPS treatment (Figure 2A), consis- tent with previous studies.63 Although the mechanism of this increase is not fully understood, studies have shown that LPS and TNF-α stimulate osteoclastogenesis and bone resorption, suggesting that, in conditions of systemic inflammation, there is a transcellular shift of phosphate from bone cells into the extracellular compartment, lead- ing to hyperphosphatemia.63,64 In response to high serum phosphate levels, circulating FGF23 and Fgf23 expression significantly increased (Figure 1D-G), followed by appro- priate decrease in renal Klotho and NaPi2a/c expression (Figure 2B-D) to increase urinary phosphate excretion (Figure 2A) in order to achieve normophosphatemia. We also investigated any potential impact of PTH on phos- phate homeostasis and we found that PTH levels are 2.5- fold increased 4 h after LPS treatment and returned to basal levels by 6 h after treatment in comparison to vehi- cle-treated mice (0 h) (data not shown), suggesting the pos- sibility that the rise in serum Pi 1-2 h after LPS may con- tribute to PTH induction, which in turn would induce phosphaturia. However, our data exclude the possibility that PTH contributes to the increase in FGF23 because the latter reaches peak levels in circulation at the same time point as PTH. Moreover, since vitamin D is a potent stim- ulator of FGF23, we measured the expression of Cyp27b1, the enzyme responsible for the synthesis of the bioactive form of vitamin D, in the kidney. We observed a 10-fold increase in renal Cyp27b1 expression 2 h after LPS treat- ment (data not shown), suggesting that circulating levels of vitamin D would be increased. However, FGF23 levels are already significantly elevated by 2 h after LPS, also exclud- ing the possibility that vitamin D contributes to the increase in FGF23. The present study cannot determine whether the increase in FGF23 levels in response to LPS is due to the rise of inflammatory cytokines such as IL-6, TNF-α and IL-1β, the decrease in serum iron, and/or the increase in serum phosphate, all occurring after LPS treat- ment and prior to the increase in FGF23 levels.
In this study we also show that, although the main source of circulating FGF23 is normally the bone, in LPS-
induced inflammation, organs involved in the immune system, such as the liver and spleen, significantly con- tribute to the increased circulating FGF23 levels (Figure 1D-G). In addition, blocking FGF23 signaling in LPS condi- tions resulted in significant reduction in bone (Figure 4A) and bone marrow (data not shown) Fgf23 mRNA levels, resulting in a modest but significant decrease in serum FGF23 levels (Figure 4B and C). However, spleen and liver Fgf23 expression were not affected by the cFGF23 in LPS- treated animals (data not shown), suggesting that these two tissues contribute to the residual induction of circulating FGF23. This is in agreement with published data showing that spleen significantly contributes to elevated circulating FGF23 levels that rise in response to acute or chronic expo- sure to LPS.65,66 Moreover, mice with genetic deletion of Fgf23 (Fgf23-/-) or its co-receptor Klotho (Klotho-/-) exhibit a reduced number of splenocytes and thymic atrophy,49,67 suggesting that Fgf23 may play a role in the regulation of the innate and/or acquired immune system. Bone marrow Fgf23 was also significantly increased between 1-12 h after LPS treatment, possibly contributing also to the increased circulating FGF23 levels (Online Supplementary Figure S1B). Fgf23 production in bone was only significantly increased after 4 h of LPS; however, this increase correlates with a similar increase in bone Fgf23 in the saline-injected group at the same time point, suggesting that it may not be LPS- dependent (Online Supplementary Figure S1A).
Moreover, in line with previously acquired knowledge that LPS-induced hypoferremia is caused through hep- cidin-dependent and -independent mechanisms,68,69 our results show that treatment of mice with LPS significantly downregulates renal Epo expression (Figure 3I) following upregulation of pro-inflammatory cytokines and Fgf23 secretion. This is consistent with studies showing that inflammatory cytokines impair erythropoiesis by inhibit- ing production of Epo, and by directly inhibiting erythroid progenitor cell proliferation and differentiation.35-37,69
The present study is the first to highlight the effect of FGF23 signaling on iron metabolism and its contribution to hypoferremia. Here, we show that at steady-state con- ditions, disruption of FGF23 signaling results in increased circulating iron levels (Figure 6A) without affecting hep- cidin (Figure 5D and E) or inflammatory status (Figure 5A- C). This result is likely due to the rise of Epo induced by the C-tail FGF23 (Figure 7D) resulting in increased splenic Fpn mRNA and protein levels (Figure 7D and Online Supplementary Figure S4). Moreover, in response to LPS, mice treated with the C-tail FGF23 resist downregulation of Hif2α, a potent inducer of Epo, resulting in increased circulating EPO and Epo expression (Figure 7). Indeed, it has been reported that Epo directly stimulates intestinal iron absorption by increasing both iron uptake through upregulation of the divalent metal transporter 1 (Dmt1) and iron efflux through upregulation of the Fpn trans- porter.70 Studies have also shown that HIF2α maintains iron balance by regulating transcription of Dmt1 and Fpn.71,72 Fpn is rapidly induced under low iron conditions via a HIF2α-dependent mechanism and disruption of HIF2α in the intestine inhibits the adaptive increase of intestinal Fpn during iron deficiency in mice.72 Together, the results of these studies suggest that HIF2α activation may induce iron mobilization, whereas inhibition of HIF2α will favor decreased iron absorption.71,72 A limita- tion of our study is the lack of data on the effect of cFGF23 on intestinal HIF2a, DMT1, and FPN protein levels, which
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