Page 78 - 2021_02-Haematologica-web

R. Agoro et al.
CKD patients have increased iron losses due to chronic bleeding from impaired intestinal iron absorption, uremia- associated platelet dysfunction, frequent phlebotomy, and blood trapping in the dialysis apparatus.10 In addition, cir- culating levels of fibroblast growth factor 23 (FGF23) dra- matically increase in CKD, reaching up to 2,000-fold above the normal range in advanced renal failure.11 Recent studies have associated iron deficiency with the increase in FGF23 levels.12,13
FGF23 has been identified as the gene responsible for the phosphate wasting disorder autosomal dominant hypophosphatemic rickets (ADHR)14 and as a causative factor of tumor-induced osteomalacia.15 FGF23 is a bone- derived endocrine regulator of phosphate and vitamin D homeostasis and bone mineralization.16-20 FGF23 inhibits phosphate reabsorption in the renal proximal tubule by suppressing the expression of the type II sodium-phos- phate transporters NaPi2a and NaPi2c in the brush border membrane.17,21-23 Furthermore, FGF23 reduces circulating levels of 1,25-dihydroxyvitamin D by suppressing the renal expression of 1α-hydroxylase,3the enzyme required for production of 1,25-dihydroxyvitamin D3, and increas- ing the activity of 24-hydroxylase, the enzyme that metabolizes 1,25-dihydroxyvitamin D3.17 FGF23 requires a co-receptor, klotho, for FGF receptor (FGFR) activation, and its effects are mediated through multiple FGFR, with FGFR1 being the principal receptor to mediate the effects of FGF23 on phosphate regulation, and FGFR3 and 4 mediating the FGF23 effects on vitamin D metabolism.24 In the kidney, FGF23 binds to the FGFR-klotho complex and induces signaling through Ras/MAPK, PI3K/Akt, and PLCγ/PKC downstream pathways which regulate mineral metabolism genes.25 FGF23 activity is regulated post-trans- lationally by proteolytic cleavage between Arg179 and Ser180, and O-linked glycosylation. Cleavage of the intact full-length FGF23 peptide by a subtilisin-like pro-protein convertase abolishes its biologic activity. Intact FGF23 contains an arginine–X–X–arginine (RXXR) motif which is recognized by pro-protein convertases such as furin. This motif is located at the boundary between the FGF core homology domain, which interacts with the FGFR, and the 72-residue-long C-terminal tail of FGF23, which inter- acts with the co-receptor Klotho.26-28
We recently reported that inhibition of FGF23 signaling rescues renal anemia in a mouse model of CKD.29 Here, we investigated the effect of inhibition of FGF23 signaling in a mouse model of LPS-induced hypoferremia. Our results show that FGF23 is induced in response to LPS prior to hepcidin upregulation followed by downregulation of Epo expression. Moreover, we show that inhibition of FGF23 signaling alleviates hypoferremia and attenuates dysregu- lation of erythropoiesis in a mouse model of acute inflam- mation induced by LPS.
Methods
Animals
C57BL/6J wild-type (WT) male mice between 8 and 10 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed at the New York University (NYU) College of Dentistry Animal Facility, where they were kept on a light/dark (12 hours [h]/12h) cycle at 23oC, and received food (standard lab chow) containing 185 parts per million iron (Teklad 2018S; Harlan) and water ad libitum. All animal protocols were approved by the
Institutional Animal Care and Use Committee (IACUC) at NYU. Mice were treated with a single intraperitoneal (i.p.) injection of 1 mg/kg of an FGF23 blocking peptide (provided by Dr. M. Mohammadi, NYU School of Medicine) or vehicle (HEPES- buffered saline; 25 mM HEPES-NaOH pH7.5, 150 mM NaCl). The FGF23 blocking peptide is the 72 aa long C-terminal tail of human FGF23 (residues Ser180-Ile251), which corresponds to the C-ter- minal fragment of FGF23 generated by proteolytic cleavage at the RXXR motif.27 Eight hours post treatment, acute inflammation was induced by a single i.p injection of 50 mg/kg of LPS (E. coli 055:B5, Sigma-Aldrich, St Louis, MO, USA). An equal volume of sterile saline solution (0.9% NaCl) was used as vehicle control. Mice were euthanized 4 h after LPS treatment. In longitudinal studies, saline- and LPS-treated mice were euthanized after 1, 2, 4, 6, 12, or 24 h of LPS treatment.
Blood, tissue, and serum collection
Mice were immediately necropsied after euthanasia and blood was collected by cardiac puncture. Complete blood count was per- formed using the VetScan HM5 Hematology Analyzer (ABAXIS, Union City, CA, USA). Serum was obtained after blood centrifu- gation at 3,500 rpm for 10 minutes (min). Liver, spleen, kidney, bones, and bone marrow cells obtained from femora and tibiae, were collected, snap-frozen in liquid nitrogen, and stored at -80°C until further use.
Serum measurements
Serum FGF23 levels were measured using the mouse FGF23 Intact and C-terminal ELISA assays (Quidel Corporation/Immutopics International, San Clemente, CA, USA). The intact assay detects exclusively biologically intact FGF23 (iFGF23), whereas the C-terminus (cFGF23) assay is capable of detecting both the intact molecule and its C-terminal fragments. Serum phosphorus was determined by colorimetric measure- ments using the Stanbio Phosphorus Liqui-UV Test reagent (Stanbio Laboratory, Boerne, TX, USA). Serum iron and transferrin saturation were measured using the Iron-TIBC kit from Pointe Scientific (Canton, MI, USA). Serum hepcidin was measured using the Hepcidin Murine-Compete ELISA kit (Intrinsic LifeSciences, La Jolla, CA, USA). Serum EPO levels were measured using the Rat/Mouse EPO Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA).
Tissue iron measurement
Hepatic and splenic iron concentration was measured by the Ferrozine colorimetric method, as described elsewhere.30,31
RNA isolation, reverse transcription, and real-time quantitative polymerase chain reaction analysis
Total RNA was extracted from kidneys, liver, whole bone, spleen, and bone marrow using Trizol (Ambion; Life Technologies, Carlsbad, CA, USA) according to the manufactur- er’s protocol (Molecular Research Center, Cincinnati, OH, USA). Synthesis of cDNA was performed using the High Capacity cDNA Reverse Transcription Kit as described by the manufacturer (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA). cDNA was amplified by quantitative polymerase chain reaction (qPCR) using the PerfecCTa SYBR Green SuperMix (Quanta Biosciences, Gaithersburg, MD, USA). All primers used in this study are listed in the Online Supplementary Table S1. mRNA levels were normalized to the housekeeping gene (Gadph or Hprt) in the same cDNA sample. The relative transcript expression of a gene is given as DCt=Cttarget−Ctreference. The fold change in gene expression, as compared to control mice, was determined as 2−DDCt values (DDCt=DCttreated−DCtcontrol).
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haematologica | 2021; 106(2)