Fibroblast Growth Factor 23 and Klotho in AKI

Summary: Acute kidney injury (AKI) is associated with many of the same mineral metabolite abnormalities that are observed in chronic kidney disease. These include increased circulating levels of the osteocyte-derived, vitamin D–regulating hormone, fibroblast growth factor 23 (FGF23), and decreased renal expression of klotho, the co-receptor for FGF23. Recent data have indicated that increased FGF23 and decreased klotho levels in the blood and urine could serve as novel predictive biomarkers of incident AKI, or as novel prognostic biomarkers of adverse outcomes in patients with established AKI. In addition, because FGF23 and klotho exert numerous classic as well as off-target effects on a variety of organ systems, targeting their dysregulation in AKI may represent a unique opportunity for therapeutic intervention. We review the pathophysiology, kinetics, and regulation of FGF23 and klotho in animal and human studies of AKI, and we discuss the challenges and opportunities involved in targeting FGF23 and klotho therapeutically. Summary: Acute kidney injury (AKI) is associated with many of the same mineral metabolite abnormalities that are observed in chronic kidney disease. These include increased circulating levels of the osteocyte-derived, vitamin D–regulating hormone, fibroblast growth factor 23 (FGF23), and decreased renal expression of klotho, the co-receptor for FGF23. Recent data have indicated that increased FGF23 and decreased klotho levels in the blood and urine could serve as novel predictive biomarkers of incident AKI, or as novel prognostic biomarkers of adverse outcomes in patients with established AKI. In addition, because FGF23 and klotho exert numerous classic as well as off-target effects on a variety of organ systems, targeting their dysregulation in AKI may represent a unique opportunity for therapeutic intervention. We review the pathophysiology, kinetics, and regulation of FGF23 and klotho in animal and human studies of AKI, and we discuss the challenges and opportunities involved in targeting FGF23 and klotho therapeutically. The osteocyte-derived hormone, fibroblast growth factor 23 (FGF23), initially was identified as the causative agent of two rare disorders of urinary phosphate wasting: autosomal-dominant hypophosphatemic rickets and tumor-induced osteomalacia.1Shimada T Mizutani S Muto T et al.Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia.Proc Natl Acad Sci U S A. 2001; 98: 6500-6505Crossref PubMed Scopus (1039) Google Scholar, 2Consortium A Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23.Nat Genet. 2000; 26: 345-348Crossref PubMed Scopus (0) Google Scholar FGF23 increases urinary phosphate excretion by reducing expression of the sodium-phosphate co-transporters, NaPi2a and NaPi2c, expressed on the apical surface of the renal proximal tubular cells, and it decreases circulating 1,25-dihydroxyvitamin D (1,25D) levels by reducing expression of the 1α-hydroxylase cytochrome P450 (CYP) enzyme, CYP27B1, which is also expressed in renal proximal tubular cells.3Shimada T Hasegawa H Yamazaki Y et al.FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.J Bone Miner Res. 2004; 19: 429-435Crossref PubMed Scopus (1081) Google Scholar FGF23 may also decrease circulating 1,25D levels by increasing the renal expression of the catabolic 24-hydroxylase enzyme, CYP24A1.3Shimada T Hasegawa H Yamazaki Y et al.FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.J Bone Miner Res. 2004; 19: 429-435Crossref PubMed Scopus (1081) Google Scholar In addition, FGF23 can increase distal sodium reabsorption by increasing the expression of the sodium chloride co-transporter, NCC, and it increases calcium absorption by increasing the expression of transient receptor potential cation channel subfamily V member 5 channels in the distal tubule.4Andrukhova O Slavic S Smorodchenko A et al.FGF23 regulates renal sodium handling and blood pressure.EMBO Mol Med. 2014; 6: 744-759Crossref PubMed Scopus (144) Google Scholar, 5Andrukhova O Smorodchenko A Egerbacher M et al.FGF23 promotes renal calcium reabsorption through the TRPV5 channel.EMBO J. 2014; 33: 229-246PubMed Google Scholar In states of excess circulating FGF23 levels and normal kidney function, humans and animals develop hyperphosphaturia, hypophosphatemia, and rickets,2Consortium A Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23.Nat Genet. 2000; 26: 345-348Crossref PubMed Scopus (0) Google Scholar, 6Yuan B Takaiwa M Clemens TL et al.Aberrant Phex function in osteoblasts and osteocytes alone underlies murine X-linked hypophosphatemia.J Clin Invest. 2008; 118: 722-734PubMed Google Scholar whereas in states of diminished FGF23 levels or function, humans and animals develop increased circulating levels of phosphate and 1,25D, and ectopic soft-tissue and vascular calcification.7Benet-Pages A Orlik P Strom TM et al.An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia.Hum Mol Genet. 2005; 14: 385-390Crossref PubMed Scopus (371) Google Scholar, 8Sitara D Razzaque MS Hesse M et al.Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice.Matrix Biol. 2004; 23: 421-432Crossref PubMed Scopus (381) Google Scholar, 9Shimada T Kakitani M Yamazaki Y et al.Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism.J Clin Invest. 2004; 113: 561-568Crossref PubMed Scopus (1023) Google Scholar FGF23 binds to single-pass transmembrane FGF receptors (FGFRs), which are expressed in a variety of tissues.10Yu X Ibrahimi OA Goetz R et al.Analysis of the biochemical mechanisms for the endocrine actions of fibroblast growth factor-23.Endocrinology. 2005; 146: 4647-4656Crossref PubMed Scopus (148) Google Scholar There are four different FGFR isoforms (FGFR1-4). In addition, alternative splicing of FGFR1-3 produces two subtypes: designated as b and c splice variants. The mineral metabolism actions of FGF23 are mediated through FGFR1, which is expressed ubiquitously.11Liu S Vierthaler L Tang W et al.FGFR3 and FGFR4 do not mediate renal effects of FGF23.J Am Soc Nephrol. 2008; 19: 2342-2350Crossref PubMed Scopus (0) Google Scholar, 12Yazaki N Fujita H Ohta M et al.The structure and expression of the FGF receptor-1 mRNA isoforms in rat tissues.Biochim Biophys Acta. 1993; 1172: 37-42Crossref PubMed Google Scholar The binding affinity of FGF23 to FGFR1 is enhanced by its co-receptor, klotho (discussed further in the “Klotho: Function and Regulators” section).13Urakawa I Yamazaki Y Shimada T et al.Klotho converts canonical FGF receptor into a specific receptor for FGF23.Nature. 2006; 444: 770-774Crossref PubMed Scopus (1157) Google Scholar It has long been thought that the differential expression of klotho, a single transmembrane domain protein expressed in the kidneys and parathyroid glands, provides the site specificity of FGF23 action. More recent work has suggested that soluble or secreted klotho can function to provide the scaffolding for FGF23 and FGFR1 binding, thus potentially expanding the number of tissues that can be targets of FGF23 action.14Chen G Liu Y Goetz R et al.alpha-klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling.Nature. 2018; 553: 461-466Crossref PubMed Scopus (90) Google Scholar In addition, FGF23 has been shown to signal through another FGF receptor, FGFR4, in the heart and liver in a klotho-independent manner.15Singh S Grabner A Yanucil C et al.Fibroblast growth factor 23 directly targets hepatocytes to promote inflammation in chronic kidney disease.Kidney Int. 2016; 90: 985-996Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 16Faul C Amaral AP Oskouei B et al.FGF23 induces left ventricular hypertrophy.J Clin Invest. 2011; 121: 4393-4408Crossref PubMed Scopus (1099) Google Scholar When FGF23 signaling occurs through FGFR1, downstream targets are affected through the MAP-kinase pathway, whereas FGF23 signaling through FGFR4 occurs via the phospholipase C Ɣ pathway. FGF23 appears to be involved in several pathways independent of mineral metabolism regulation, and its list of off-target effects is still growing. FGF23 consists of 251 amino acids, including a 24 amino acid signal peptide. FGF23 production is increased by high blood phosphate levels or dietary phosphate loading, 1,25D, and numerous other stimuli (Fig. 1). Most studies found that parathyroid hormone (PTH) increased FGF23 production (reviewed by Bergwitz and Jüppner17Bergwitz C Jüppner H Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23.Annu Rev Med. 2010; 61: 91-104Crossref PubMed Scopus (336) Google Scholar), although some studies found that acute PTH treatment initially may decrease circulating FGF23 levels in both humans and mice.18Knab VM Corbin B Andrukhova O et al.Acute parathyroid hormone injection increases C-terminal but not intact fibroblast growth factor 23 levels.Endocrinology. 2017; 158: 1130-1139Crossref PubMed Scopus (6) Google Scholar, 19Gutierrez OM Smith KT Barchi-Chung A et al.(1-34) Parathyroid hormone infusion acutely lowers fibroblast growth factor 23 concentrations in adult volunteers.Clin J Am Soc Nephrol. 2012; 7: 139-145Crossref PubMed Scopus (0) Google Scholar Recent reports have implicated iron deficiency and hypoxia as additional factors that up-regulate FGF23 production; however, the underlying mechanisms remain incompletely understood.20Farrow EG Yu X Summers LJ et al.Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice.Proc Natl Acad Sci U S A. 2011; 108: E1146-E1155Crossref PubMed Scopus (195) Google Scholar, 21Clinkenbeard EL Farrow EG Summers LJ et al.Neonatal iron deficiency causes abnormal phosphate metabolism by elevating FGF23 in normal and ADHR mice.J Bone Miner Res. 2014; 29: 361-369Crossref PubMed Scopus (60) Google Scholar Chronic inflammation, possibly through an iron-deficiency/hypoxia axis, also increases FGF23 production.22David V Martin A Isakova T et al.Inflammation and functional iron deficiency regulate fibroblast growth factor 23 production.Kidney Int. 2016; 89: 135-146Abstract Full Text Full Text PDF PubMed Google Scholar Both oral and intravenous iron supplementation have been shown to reduce plasma FGF23 levels in patients with iron-deficiency anemia in the setting of both normal and reduced renal function.23Wolf M Koch TA Bregman DB Effects of iron deficiency anemia and its treatment on fibroblast growth factor 23 and phosphate homeostasis in women.J Bone Miner Res. 2013; 28: 1793-1803Crossref PubMed Scopus (165) Google Scholar, 24Iguchi A Kazama JJ Yamamoto S et al.Administration of ferric citrate hydrate decreases circulating FGF23 levels independently of serum phosphate levels in hemodialysis patients with iron deficiency.Nephron. 2015; 131: 161-166Crossref PubMed Scopus (28) Google Scholar Early studies have shown Fgf23 messenger RNA (mRNA) expression in the heart, liver, thyroid/parathyroid, small intestine, skeletal muscle, and fetal chondrocytes, as well as lymph nodes.1Shimada T Mizutani S Muto T et al.Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia.Proc Natl Acad Sci U S A. 2001; 98: 6500-6505Crossref PubMed Scopus (1039) Google Scholar, 2Consortium A Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23.Nat Genet. 2000; 26: 345-348Crossref PubMed Scopus (0) Google Scholar Subsequently, Fgf23 expression was documented in osteocytes, especially in the case of animals or humans with chronic kidney disease (CKD), where osteocytes are believed to be the major source of increased circulating FGF23 levels.25Pereira RC Juppner H Azucena-Serrano CE et al.Patterns of FGF-23, DMP1, and MEPE expression in patients with chronic kidney disease.Bone. 2009; 45: 1161-1168Crossref PubMed Scopus (178) Google Scholar Recent work also has shown Fgf23 mRNA expression in liver, kidneys, heart, spleen, and bone marrow.26Prie D Forand A Francoz C et al.Plasma fibroblast growth factor 23 concentration is increased and predicts mortality in patients on the liver-transplant waiting list.PLoS One. 2013; 8: e66182Crossref PubMed Scopus (0) Google Scholar, 27Bansal S Friedrichs WE Velagapudi C et al.Spleen contributes significantly to increased circulating levels of fibroblast growth factor23 in response to lipopolysaccharide-induced inflammation.Nephrol Dial Transplant. 2017; 32: 583Crossref PubMed Scopus (0) Google Scholar, 28Andrukhova O Slavic S Odorfer KI et al.Experimental myocardial infarction upregulates circulating fibroblast growth factor-23.J Bone Miner Res. 2015; 30: 1831-1839Crossref PubMed Scopus (39) Google Scholar, 29Clinkenbeard EL Hanudel MR Stayrook KR et al.Erythropoietin stimulates murine and human fibroblast growth factor-23, revealing novel roles for bone and bone marrow.Haematologica. 2017; 102: e427-e430Crossref PubMed Scopus (30) Google Scholar, 30Zanchi C Locatelli M Benigni A et al.Renal expression of FGF23 in progressive renal disease of diabetes and the effect of ACE inhibitor.PLoS One. 2013; 8: e70775Crossref PubMed Scopus (62) Google Scholar An important step in the regulation of FGF23 metabolism is post-translational modification of the cleavage of bioactive intact FGF23 into C-terminal and N-terminal fragments (Fig. 2). FGF23 undergoes cleavage between arginine 179 and serine 180, located in a conserved cleavage site R176XXR179. Cleavage of FGF23 at this site is down-regulated by O-glycosylation of tyrosine residue 178.31Kato K Jeanneau C Tarp MA et al.Polypeptide GalNAc-transferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O-glycosylation.J Biol Chem. 2006; 281: 18370-18377Crossref PubMed Scopus (265) Google Scholar Cleavage at the RXXR site generates a C-terminal 12-kDa and an N-terminal 18-kDa fragment from the 30- to 32-kDa intact, glycosylated FGF23 protein. Additional post-translational modification of FGF23 occurs by phosphorylation of serine 180, adjacent to the RXXR cleavage site, through the kinase FAM20C. Phosphorylation of this residue inhibits glycosylation, thus enhancing FGF23 cleavage (Fig. 2).32Tagliabracci VS Engel JL Wiley SE et al.Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis.Proc Natl Acad Sci U S A. 2014; 111: 5520-5525Crossref PubMed Scopus (96) Google Scholar Circulating FGF23 levels comprise the bioactive intact hormone (iFGF23) and N-terminal and C-terminal fragments, although at physiological concentrations the hormonal fragments likely do not contribute to mineral ion regulation. At supraphysiological concentrations the C-terminal fragment can interfere with FGF23 action, both in the kidney and in the bone marrow, by competing with iFGF23 for the FGF23 receptor.33Goetz R Nakada Y Hu MC et al.Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-klotho complex formation.Proc Natl Acad Sci U S A. 2010; 107: 407-412Crossref PubMed Scopus (180) Google Scholar, 34Agoro R Montagna A Goetz R et al.Inhibition of fibroblast growth factor 23 (FGF23) signaling rescues renal anemia.FASEB J. 2018; 32: 3752-3764Crossref PubMed Scopus (13) Google Scholar However, in a fibroblast cell line, C-terminal FGF23 did not show any biological activity.35Smith ER Tan SJ Holt SG et al.FGF23 is synthesised locally by renal tubules and activates injury-primed fibroblasts.Sci Rep. 2017; 7: 3345Crossref PubMed Scopus (27) Google Scholar In states of iron deficiency and acute inflammation, there was increased production of FGF23, which was matched by a commensurate (or near-commensurate) increase in FGF23 cleavage, such that there are high levels of FGF23 fragments in the circulation but normal (or only mildly increased) levels of iFGF23. In contrast, in CKD and end-stage renal disease (ESRD) there is a presumed down-regulation of the cleavage mechanism, and iFGF23 accounts for a larger proportion of the total circulating levels of FGF23.36Shimada T Urakawa I Isakova T et al.Circulating fibroblast growth factor 23 in patients with end-stage renal disease treated by peritoneal dialysis is intact and biologically active.J Clin Endocrinol Metab. 2010; 95: 578-585Crossref PubMed Scopus (147) Google Scholar, 37Smith ER Cai MM McMahon LP et al.Biological variability of plasma intact and C-terminal FGF23 measurements.J Clin Endocrinol Metab. 2012; 97: 3357-3365Crossref PubMed Scopus (101) Google Scholar Two main assays are used to measure circulating FGF23 levels. The immunometric assay for C-terminal FGF23 (cFGF23) detects both C-terminal cleavage fragments as well as the full-length intact peptide, whereas the assay for iFGF23 measures intact FGF23 only. The primary mode of clearance of FGF23 and its fragments from the circulation is not well understood, although several groups have reported that FGF23 is detectable in the urine.38Jonsson KB Zahradnik R Larsson T et al.Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia.N Engl J Med. 2003; 348: 1656-1663Crossref PubMed Scopus (634) Google Scholar, 39Leaf DE Jacob KA Srivastava A et al.Fibroblast growth factor 23 levels associate with AKI and death in critical illness.J Am Soc Nephrol. 2017; 28: 1877-1885Crossref PubMed Scopus (30) Google Scholar, 40Wesseling-Perry K Pereira RC Tsai E et al.FGF23 and mineral metabolism in the early post-renal transplantation period.Pediatr Nephrol. 2013; 28: 2207-2215Crossref PubMed Scopus (19) Google Scholar Mace et al41Mace ML Gravesen E Nordholm A et al.Kidney fibroblast growth factor 23 does not contribute to elevation of its circulating levels in uremia.Kidney Int. 2017; 92: 165-178Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar measured differences in intact FGF23 between the renal artery and vein of rats, and reported that the extraction ratio was 0.3, indicating that iFGF23 levels in the vein were 30% lower compared with levels in the artery. These findings are consistent with a report in patients with CKD, in which the extraction of FGF23 was approximately 17%, although varied by renal function, with higher extraction in patients with higher glomerular filtration rates.42van Ballegooijen AJ Rhee EP Elmariah S et al.Renal clearance of mineral metabolism biomarkers.J Am Soc Nephrol. 2016; 27: 392-397Crossref PubMed Google Scholar These findings suggest that the kidneys participate in clearance of FGF23, either through filtration and/or catabolism. Accordingly, this mode of clearance may be impaired in patients with acutely or chronically reduced kidney function. 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