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      Upregulation of the Creatine Transporter Slc6A8 by Klotho

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          Background/Aims: The transmembrane Klotho protein contributes to inhibition of 1,25(OH)<sub>2</sub>D<sub>3</sub> formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. β-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8), which participates in the maintenance of neuronal function and survival. Methods: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8) activity was estimated from creatine induced current determined by two-electrode voltage-clamp. Results: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM). The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by β-glucuronidase inhibitor (DSAL). Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. Conclusion: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8) by stabilizing the carrier protein in the cell membrane, an effect requiring β-glucuronidase activity of Klotho protein.

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          Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.

          Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi-2a. The phosphaturic effect was present in FGF23-null mice, indicating a direct action distinct from Klotho's known role as a coreceptor for FGF23. Direct inhibition of NaPi-2a by Klotho was confirmed in cultured cells and in cell-free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant beta-glucuronidase and is associated with proteolytic degradation and reduced surface NaPi-2a. The inhibitory effect of Klotho on NaPi-2a was blocked by beta-glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi-2a from the apical membrane.
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            Secreted Klotho protein in sera and CSF: implication for post-translational cleavage in release of Klotho protein from cell membrane.

            Klotho mutant mice exhibit a set of phenotypes resembling human ageing. Although the function of Klotho remains unclear, mediation of its pleiotropic functions by putative humoral factor(s) has been presumed. Newly established antibodies against Klotho allowed the detection of secreted Klotho, a candidate for the putative humoral factor, in sera and cerebrospinal fluid. Surprisingly the secreted Klotho was 130 kDa, in contrast to the 70 kDa predicted form from klotho gene transcripts. The secreted as well as the membrane-bound Klotho proteins were suggested to form oligomerized complex. These results delineate post-translation processing of Klotho and possible regulatory mechanisms for secretion of Klotho in vivo.
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              Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1.

              Klotho is a mammalian senescence-suppression protein that has homology with glycosidases. The extracellular domain of Klotho is secreted into urine and blood and may function as a humoral factor. Klotho-deficient mice have accelerated aging and imbalance of ion homeostasis. Klotho treatment increases cell-surface abundance of the renal epithelial Ca(2+) channel TRPV5 by modifying its N-linked glycans. However, the precise sugar substrate and mechanism for regulation by Klotho is not known. Here, we report that the extracellular domain of Klotho activates plasma-membrane resident TRPV5 through removing terminal sialic acids from their glycan chains. Removal of sialic acids exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 lattice at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. Knockdown of beta-galactoside alpha2,6-sialyltransferase (ST6Gal-1) by RNA interference, but not other sialyltransferases, in a human cell line prevents the regulation by Klotho. Moreover, the regulation by Klotho is absent in a hamster cell line that lacks endogenous ST6Gal-1, but is restored by forced expression of recombinant ST6Gal-1. Thus, Klotho participates in specific removal of alpha2,6-linked sialic acids and regulates cell surface retention of TRPV5 through this activity. This action of Klotho represents a novel mechanism for regulation of the activity of cell-surface glycoproteins and likely contributes to maintenance of calcium balance by Klotho.

                Author and article information

                Kidney Blood Press Res
                Kidney and Blood Pressure Research
                S. Karger AG
                December 2014
                29 November 2014
                : 39
                : 6
                : 516-525
                aDepartment of Physiology, Gmelinstr. 5, University of Tübingen, D-72076 Tübingen, Germany; bDepartment of Psychiatry, University Hospital Basel, Switzerland; cFaculty of Medicine, University of Prishtina, Str. Bulevardi i Dëshmorëve; dDepartment of Chemistry, University of Prishtina, Str. 'Nëna Terezë' p.n. 10 000 Prishtina, Kosova
                Author notes
                *Florian Lang, MD, Department of Physiology, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, (Germany), Tel. +49-70712972194, Fax +49-7071295618, E-Mail florian.lang@uni-tuebingen.de
                368462 Kidney Blood Press Res 2014;39:516-525
                © 2014 S. Karger AG, Basel

                Open Access License: This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) ( http://www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Pages: 10
                Original Paper

                Cardiovascular Medicine, Nephrology

                Creatine, CreaT, β-glucuronidase, DSAL, Brain


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