Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease
Authors: Christopher Yanucil, Dominik Kentrup, Isaac Campos, Brian Czaya, Kylie Heitman, David Westbrook, Gunars Osis, Alexander Grabner, Adam R. Wende, Julian Vallejo, Michael J. Wacker, Jose Alberto Navarro-Garcia, Gema Ruiz-Hurtado, Fuming Zhang, Yuefan Song, Robert J. Linhardt, Kenneth White, Michael S. Kapiloff, Christian Faul
Journal: Kidney International (2022)

Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied. Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied. Translational StatementPatients on hemodialysis have extremely high fibroblast growth factor 23 (FGF23) levels, and during dialysis, they receive infusions of heparin as a blood thinner. Our experimental studies show that heparin acts as a cofactor that increases FGF23's binding affinity for the cardiac FGF23 receptor and thereby aggravates the pathologic actions of FGF23 on the heart. Hemodialysis does not reduce the cardiovascular risk of patients with end-stage renal disease, and future clinical studies should test whether frequent heparin infusions contribute to the extremely high mortality rates in these patients and whether removal of heparin from the dialysis process might improve cardiac outcomes and survival. Fibroblast growth factor (FGF) 23 is a hormone that serves as a major regulator of phosphate metabolism.1Quarles L.D. Endocrine functions of bone in mineral metabolism regulation.J Clin Invest. 2008; 118: 3820-3828Crossref PubMed Scopus (362) Google Scholar In response to elevations in serum phosphate levels, such as after food intake, FGF23 is secreted by the bone and targets the kidney via FGF receptor (FGFR) isoform 1c (FGFR1c) and the coreceptor α-klotho (called "klotho" from here on), resulting in the induction of Ras/mitogen-activated protein kinase (MAPK) signaling.2Kuro-o M. Matsumura Y. 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Aizawa H. et al.Mutation of the mouse klotho gene leads to a syndrome resembling ageing.Nature. 1997; 390: 45-51Crossref PubMed Scopus (2755) Google Scholar,13Faul C. Amaral A.P. Oskouei B. et al.FGF23 induces left ventricular hypertrophy.J Clin Invest. 2011; 121: 4393-4408Crossref PubMed Scopus (1463) Google Scholar,54Hu M.C. Shi M. Cho H.J. et al.Klotho and phosphate are modulators of pathologic uremic cardiac remodeling.J Am Soc Nephrol. 2015; 26: 1290-1302Crossref PubMed Scopus (197) Google Scholar,55Hu M.C. Shi M. Zhang J. et al.Klotho deficiency causes vascular calcification in chronic kidney disease.J Am Soc Nephrol. 2011; 22: 124-136Crossref PubMed Scopus (695) Google Scholar Furthermore, klotho deficiency predisposes the heart to stress-induced cardiac injury.56Yang K. Wang C. Nie L. et al.Klotho protects against indoxyl sulphate-induced myocardial hypertrophy.J Am Soc Nephrol. 2015; 26: 2434-2446Crossref PubMed Scopus (125) Google Scholar,57Xie J. Cha S.K. 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Shi M. Zhang J. et al.Klotho deficiency causes vascular calcification in chronic kidney disease.J Am Soc Nephrol. 2011; 22: 124-136Crossref PubMed Scopus (695) Google Scholar,58Xie J. Yoon J. An S.W. et al.Soluble klotho protects against uremic cardiomyopathy independently of fibroblast growth factor 23 and phosphate.J Am Soc Nephrol. 2015; 26: 1150-1160Crossref PubMed Scopus (173) Google Scholar it is assumed that a reduction in sKL's direct protective effects on the cardiovascular system causes injury. However, experimental evidence indicating that sKL protects tissues by directly targeting them is still missing. It remains unclear whether pathologies associated with a global reduction in klotho expression are caused indirectly by the loss of transmembrane klotho in the kidney, resulting in systemic alterations, such as elevations in serum levels of phosphate or FGF23, or directly by the absence of sKL and its tissue protective actions. To date, an sKL receptor has not been identified and it remains unclear whether sKL can directly induce signal transduction and modulate effects in specific target cells such as cardiac myocytes.59Grabner A. Faul C. The role of fibroblast growth factor 23 and Klotho in uremic cardiomyopathy.Curr Opin Nephrol Hypertens. 2016; 25: 314-324Crossref PubMed Scopus (40) Google Scholar It has been postulated that the direct cellular effects of sKL require intrinsic glycosidase activity60Hu M.C. Shi M. Zhang J. et al.Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.FASEB J. 2010; 24: 3438-3450Crossref PubMed Scopus (465) Google Scholar, 61Chang Q. Hoefs S. van der Kemp A.W. et al.The β-glucuronidase klotho hydrolyzes and activates the TRPV5 channel.Science. 2005; 310: 490-493Crossref PubMed Scopus (515) Google Scholar, 62Cha S.K. Ortega B. 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New insights into the mechanism of action of soluble klotho.Front Endocrinol (Lausanne). 2017; 8: 323Crossref PubMed Scopus (98) Google Scholar The recent analysis of the crystal structure revealed that FGF23, sKL, and FGFR1c form a complex, where sKL functions as a nonenzymatic scaffold protein that brings FGFR1c and FGF23 in close proximity, thereby conferring stability of the ternary complex and promoting FGF23-mediated signaling.65Chen G. Liu Y. Goetz R. et al.α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling.Nature. 2018; 553: 461-466Crossref PubMed Scopus (235) Google Scholar This study suggests that sKL acts as an on-demand bona fide coreceptor for FGF23 and that the effects of sKL are FGF23 dependent, thereby challenging the concept that sKL can function as an FGF23-independent hormone. Surprisingly, this structural study also revealed the presence of HS in the FGF23/FGFR1c/sKL complex.65Chen G. Liu Y. Goetz R. et al.α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling.Nature. 2018; 553: 461-466Crossref PubMed Scopus (235) Google Scholar sKL and HS are part of the FGF23/FGFR signaling complex, and here we study the order of binding events among the different components. We determine whether sKL and HS function as independent FGF23 coreceptors and act on different FGFR isoforms, and we analyze whether both cofactors modulate the pathologic FGFR4-mediated actions of FGF23 on the heart. Mouse soluble and full-length klotho and β-klotho cDNAs were kindly provided by Dr. Makoto Kuro-o (University of Texas Southwestern Medical Center, Dallas, TX),43Kurosu H. Ogawa Y. Miyoshi M. et al.Regulation of fibroblast growth factor-23 signaling by klotho.J Biol Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1058) Google Scholar,66Ogawa Y. Kurosu H. Yamamoto M. et al.βKlotho is required for metabolic activity of fibroblast growth factor 21.Proc Natl Acad Sci U S A. 2007; 104: 7432-7437Crossref PubMed Scopus (471) Google Scholar and human soluble and full-length klotho cDNA constructs were from Amgen.67Shi S.Y. Lu Y.W. Liu Z. et al.A biparatopic agonistic antibody that mimics fibroblast growth factor 21 ligand activity.J Biol Chem. 2018; 293: 5909-5919Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Inserts were subcloned into vectors with appropriate epitope tags, that is, C-terminal Strep/6xHis (OG1240, Oxford Genetics) or C-terminal FLAG/His (OG1232, Oxford Genetics). Subcloning was performed with appropriate restriction enzymes (New England Biolabs [NEB]) according to the manufacturer's protocols, and constructs were verified by Sanger sequencing. The EGFP-N1 vector used for green fluorescent protein (GFP) overexpression was from Clontech. Recombinant proteins from R&D Systems are human FGF2 (233-FB/CF), human FGF5 (237-F5/CF), human FGF7 (251-KG/CF), human FGF8b (423-F8/CF), human FGF19 (969FG025/CF), human FGF21 (2539FG025/CF), human FGF23 (2604FG025/CF), mouse FGF23 (2629FG025/CF), human FGFR1c (658FR050), human FGFR2c (712FR050), human FGFR3c (766FR050), and human FGFR4 (685FR050). The sKL recombinant protein produced by our laboratory is mouse aa 35-982 and human aa 34-981. Mouse sKL from R&D Systems (1819KL050) is aa 35-982; human klotho domain 1 (KL1) from PeproTech is aa 34-549. Heparin solution is from Celsus Laboratories for surface plasmon resonance experiments and from Pfizer Injectables for all other studies (NDC0069005902). Primary antibodies used are anti-FGF19 (AF969, R&D Systems), anti-FGF21 (AF2539, R&D Systems), anti-FGF23 (AF2604, R&D Systems), anti-FLAG (F1804, Sigma-Aldrich), ant