title: Membrane-bound Klotho is not expressed endogenously in healthy or uraemic human vascular tissue.
abstract: OBJECTIVE
  Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD), a disease state that is strongly associated with loss of renal and systemic (alpha-)Klotho. Reversely, murine Klotho deficiency causes marked medial calcification. It is therefore thought that Klotho conveys a vasculoprotective effect. Klotho expression in the vessel wall, however, is disputed.
  
  
  RESULTS
  We assessed Klotho expression in healthy human renal donor arteries (n = 9), CKD (renal graft recipient) arteries (n = 10), carotid endarterectomy specimens (n = 8), other elastic arteries (three groups of n = 3), and cultured human aortic smooth muscle cells (HASMCs) (three primary cell lines), using immunohistochemistry (IHC), immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, and western blotting (WB). We have extensively validated anti-Klotho antibody KM2076 by comparing staining patterns with other anti-Klotho antibodies (SC-22220, SC-22218, and AF1819), competition assays with recombinant Klotho, IHC on Klotho-deficient kl/kl mouse kidney, and WB with recombinant Klotho. Using KM2076, we could not detect full-length Klotho in vascular tissues or HASMCs. On the mRNA level, using primers against all four exon junctions, klotho expression could not be detected either. Fibroblast growth factor 23 (FGF23) injections in mice induced FGF23 signalling in kidneys but not in the aorta, indicating the absence of Klotho-dependent FGF23 signalling in the aorta.
  
  
  CONCLUSIONS
  Using several independent and validated methods, we conclude that full-length, membrane-bound Klotho is not expressed in healthy or uraemic human vascular tissue.
authors.affiliation: Department of Pathology and Medical Biology (Division of Pathology), University of Groningen, University Medical Center Groningen, HPC EA10, PO Box 30.001, Groningen 9700 RB, The Netherlands.
authors.forename: Rik
authors.initials: R
authors.lastname: Mencke
authors.affiliation: Department of Pathology and Medical Biology (Division of Pathology), University of Groningen, University Medical Center Groningen, HPC EA10, PO Box 30.001, Groningen 9700 RB, The Netherlands.
authors.forename: Geert
authors.initials: G
authors.lastname: Harms
authors.affiliation: Department of Internal Medicine (Division of Nephrology), University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Katarina
authors.initials: K
authors.lastname: Mirković
authors.affiliation: Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Joyce
authors.initials: J
authors.lastname: Struik
authors.affiliation: Department of Pathology and Medical Biology (Division of Pathology), University of Groningen, University Medical Center Groningen, HPC EA10, PO Box 30.001, Groningen 9700 RB, The Netherlands.
authors.forename: Joris
authors.initials: J
authors.lastname: Van Ark
authors.affiliation: Department of Physiology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Ellen
authors.initials: E
authors.lastname: Van Loon
authors.affiliation: Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Melissa
authors.initials: M
authors.lastname: Verkaik
authors.affiliation: Department of Internal Medicine (Division of Nephrology), University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Martin H
authors.initials: MH
authors.lastname: De Borst
authors.affiliation: Department of Surgery (Division of Vascular Surgery), University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Clark J
authors.initials: CJ
authors.lastname: Zeebregts
authors.affiliation: Department of Physiology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Joost G
authors.initials: JG
authors.lastname: Hoenderop
authors.affiliation: Department of Nephrology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Marc G
authors.initials: MG
authors.lastname: Vervloet
authors.affiliation: Department of Pathology and Medical Biology (Division of Pathology), University of Groningen, University Medical Center Groningen, HPC EA10, PO Box 30.001, Groningen 9700 RB, The Netherlands j.l.hillebrands@umcg.nl.
authors.forename: Jan-Luuk
authors.initials: JL
authors.lastname: Hillebrands
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007083:Iliac Artery / Q000378:metabolism; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism*; D007676:Kidney Failure, Chronic / Q000378:metabolism; D016030:Kidney Transplantation; D000090265:Klotho Proteins; D051379:Mice; D008875:Middle Aged; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D012077:Renal Artery / Q000378:metabolism*; D014511:Uremia / Q000378:metabolism; D055815:Young Adult
chemical_list: C000717427:FGF23 protein, human; C000717428:Fgf23 protein, mouse; D005346:Fibroblast Growth Factors; D000089703:Fibroblast Growth Factor-23; D005966:Glucuronidase; D000090265:Klotho Proteins
keywords: Cardiovascular disease; Chronic kidney disease; Fibroblast growth factor 23; Klotho; Smooth muscle cells