University of Groningen
Klotho in vascular biology
Mencke, Rik
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Mencke, R. (2018). Klotho in vascular biology. Rijksuniversiteit Groningen.
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Klotho in Vascular Biology
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© Rik Mencke, 2018
All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without permission of the author. Cover Design and Art Work: Spinning the thread of life, Ineke Jansen
Book Design: Rik Mencke
Print: GVO drukkers en vormgevers B.V. ISBN (printed): 978-94-034-1025-8
ISBN (digital): 978-94-034-1024-1 This PhD project was financially supported by: University Medical Center Groningen
Groningen University Institute for Drug Exploration
Junior Scientific Masterclass, Faculty of Medicine, University of Groningen Dutch Kidney Foundation
Jan Kornelis de Cock Foundation
The printing of this thesis was kindly supported by: University Medical Center Groningen
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Klotho in Vascular Biology
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op maandag 19 november 2018 om 11.00 uur
door
Rik Mencke
geboren op 1 augustus 1990 te Emmen
4 Promotor Prof. dr. J.L. Hillebrands Copromotor Dr. M.G. Vervloet Beoordelingscommissie Prof. dr. G. Molema Prof. dr. S.P. Berger Prof. dr. L. Schurgers
5 Paranimfen
L.F.A. van Dullemen W.T. van Haaften
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Table of Contents
Chapter 1 General introduction page 9
Chapter 2 The role of the anti-ageing protein Klotho in vascular page 21 physiology and pathophysiology
Part I Vascular and renal Klotho expression page 81
Chapter 3 Tissue expression and source of circulating αKlotho page 83
Chapter 4 Membrane-bound Klotho is not expressed endogenously in page 133 healthy or uremic human vascular tissue
Chapter 5 Assessment of vascular Klotho expression and functionality page 165
Chapter 6 Human alternative Klotho mRNA is a nonsense-mediated page 191 mRNA decay target inefficiently spliced in renal disease
Part II Klotho and arterial remodeling page 231
Chapter 7 Imaging of incipient vascular calcification in Klotho deficiency page 233
Chapter 8 Klotho deficiency induces arteriolar hyalinosis in a trade-off page 259 with vascular calcification
Chapter 9 Klotho deficiency promotes and induces the development of page 285 intimal hyperplasia
Chapter 10 Characterization of vascular function in Klotho deficiency page 309
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Part III Towards clinical applications of Klotho page 355
Chapter 12 Effects of Klotho on fibrosis and cancer: a renal focus on page 357 mechanisms and therapeutic strategies
Chapter 13 Clinical aspects of Klotho in vascular disease page 401
Chapter 14 Association of Klotho allele variants with graft survival page 437 after kidney transplantation
Chapter 15 General discussion and future perspectives page 451
Appendices page 465
Nederlandse samenvatting page 467
Acknowledgments page 475
Author affiliations page 483
Publications page 489
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Chapter 1
General introduction
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The protein Klotho was first described in 1997, after the serendipitous generation of a mouse with a disruption in the promoter of an unknown gene (1). This new knockout mouse was found to develop a systemic syndrome resembling human ageing and re-introduction of the gene reversed the ageing phenotype, serving as the inspiration for naming the gene and protein Klotho, for the eponymous mythological Greek goddess Κλωθώ who was thought to determine lifespan by spinning the thread of life. It was later determined that Klotho overexpression indeed had the opposite effect and extended lifespan in mice by 20-30% (2).
Klotho deficiency and overexpression
Deficiency of Klotho induces a premature ageing-like syndrome that includes a short lifespan (1), vascular calcification (3, 4), osteoporosis (5), pulmonary emphysema (6, 7), cardiac hypertrophy (8, 9), a decrease in renal function (10), cognitive dysfunction (11, 12), infertility (1), hearing deficits (13), decrease in retinal function (14), and general atrophy of muscles (15), skin (16), and other tissues (1). This is a remarkably extensive ageing phenotype for a single gene deficiency. Conversely, overexpression or supplementation of Klotho protects against renal disease (17-29), cardiac disease (8, 17, 30-34), pulmonary disease (35, 36), neurodegenerative disease (37-42), muscle disease (27, 43, 44), diabetes (45, 46), and various tumors (22, 47, 48).
Figure 1. Paradigm of Klotho protein expression. (A) Schematic overview: mRNA for membrane-bound Klotho and an alternatively spliced Klotho mRNA transcripts are transcribed. The normal transcript is known to code for the membrane-bound Klotho protein, containing KL1 and KL2 regions and two sites for proteolytic cleavage, which generate full-length soluble Klotho and separate KL1 and KL2 domains. Secreted Klotho is thought to be translated as a splice variant. (B) Klotho protein expression pattern in human kidney, using antibody KM2076. Original magnification 320×.
11 Klotho and chronic kidney disease (CKD)
Membrane-bound Klotho is expressed primarily in the distal convoluted tubule in the kidney (with additional expression in the choroid plexus, parathyroid gland, and sinoatrial node) and contains two internally homologous regions termed KL1 and KL2 (1, 49). Klotho is also cleaved off of the membrane, generating soluble Klotho, which is found in blood, urine, and cerebrospinal fluid (50-53). It has also long been hypothesized that a putative shorter Klotho protein, termed secreted Klotho, would be the product of alternative splicing (54). Figure 1A displays the paradigm of forms of Klotho proteins. The fact that Klotho is predominantly expressed in the distal convoluted tubule in the kidney (Figure 1B) explains the early occurrence of Klotho deficiency in chronic kidney disease (CKD) (10, 55, 56). Since patients with CKD also develop a premature ageing-like phenotype (57), the question is raised to what extent lack of Klotho is responsible for that. More specifically, the extensive mineral homeostasis imbalances that develop in CKD-mineral bone disorder (MDB) are very similar to the phenotype of Klotho knockout mice. This includes hyperphosphatemia, which develops as a consequence of ablated fibroblast growth factor 23 (FGF23) signaling in the absence of Klotho as an obligate co-receptor for FGFR1c, thereby reducing phosphaturia and contributing to the development of vascular calcification (58, 59). The prominence of vascular calcification in Klotho deficiency points to Klotho being a major player in cross-talk between the kidney and the cardiovascular system. The excessive cardiovascular mortality in CKD patients (60) raises the question whether maintenance of Klotho levels would be beneficial in preventing cardiovascular disease in these patients.
Klotho and the vasculature
The vascular effects of various degrees of Klotho deficiency, which include vascular calcification (1), endothelial dysfunction (61, 62), arterial stiffening (63, 64), impaired angiogenesis (65), and hypertension (66, 67), are increasingly being recognized as potentially relevant to both ageing and vascular complications of CKD. Klotho overexpression and supplementation have so far been shown to protect against vascular calcification (4, 10), endothelial dysfunction (68), atherosclerosis (69), thrombosis (69), and hypertension (28). These effects are at least in part mediated by soluble Klotho, but there are a lot of contradictory data on whether Klotho may be expressed in the vasculature itself (70, 71). Whether the aforementioned anti-tumor effects are in part due to Klotho effects on tumor angiogenesis is also unknown. Overall, despite significant gaps in our current knowledge, Klotho appears to be a promising target in designing novel therapies for ageing-related and CKD-related vascular disease.
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Aim and scope of this thesis
The aim of this thesis is to investigate the role of Klotho in vascular biology, with a particular focus on vascular Klotho expression and the role of Klotho in vascular remodeling, smooth muscle cell (SMC) de-differentiation and associated pathological SMC behavior. Chapter 2
provides a broad overview of what was previously known about the link between Klotho and the vasculature, describing the vascular Klotho deficiency phenotype, interventions that have been performed to modulate the Klotho deficiency phenotype, and the experimental effects of increased Klotho levels. We also provide a synthesis of the data on vascular Klotho expression, and describe what is known about the mechanisms in which Klotho affects SMCs and endothelial cells (ECs).
Part I then focuses on renal and vascular Klotho expression. In Chapter 3, we review the
current knowledge on Klotho expression, with a special emphasis on providing a comparative framework for tissues/cell types, a classification for anti-Klotho antibody validation, the controversy surrounding vascular Klotho expression, and the establishment of the kidney as the principal source for circulating, soluble Klotho. In Chapter 4, we investigate whether
membrane-bound Klotho is expressed in human arterial tissue, with particular emphasis on
Klotho protein expression and antibody validation. In Chapter 5, we continue our
investigation of vascular Klotho expression, further addressing the controversy around vascular immunoreactivity of anti-Klotho antibodies. We also investigate whether artery-specific Klotho knockout mice have a vascular phenotype. Chapter 6, in turn, focuses more
generally on the concept of secreted Klotho, which has long been thought to be the product of an alternatively spliced Klotho mRNA transcript and of which we study the translation and potential clinical relevance.
Part II centers around the role of Klotho in arterial remodeling with a particular focus on
aberrant SMC behavior. In Chapter 7, we investigate the development of vascular calcification
in Klotho deficiency using various imaging techniques and we assess whether new insights in the pathophysiology of vascular calcification in CKD with regard to calciprotein particles are also applicable to Klotho deficiency-induced vascular calcification. In Chapter 8, we detail that
Klotho-deficient mice are also affected by arteriolar hyalinosis, which is also seen in the kidney in human ageing, and we investigate the phenotypic variability of Klotho deficiency using different Klotho knockout strains. Chapter 9 then focuses on whether Klotho deficiency leads
to or exacerbates intimal hyperplasia, which was originally thought to be the case, but which has not been investigated since. To this end, we used different experimental models of intimal injury, comparing Klotho+/- and WT mice. In Chapter 10, we study vascular function in Klotho
deficiency ex vivo, both focusing on endothelial dysfunction and SMC contractility. Continuing our shift of focus towards the endothelium, we describe in Chapter 11 our experiments on
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example of a highly-angiogenic tumor. While anti-tumor effects of Klotho are well-recorded in various tumors, this is the first study assessing the effect of Klotho on tumor angiogenesis.
Part III is a collection of clinically oriented chapters, aimed at working towards clinical
applications of Klotho in diagnostics or therapy. Chapter 12 describes broadly how Klotho has
been used experimentally in animal models of fibrosis and cancer, which reveals how, so far, pre-clinical evidence towards a potential therapy has been supported by lines of evidence narrowing down how and which forms of Klotho can be used. Furthermore, we describe what we now about Klotho structure-function relationships in various pathways, which therapeutic
strategies have been attempted in delivering Klotho experimentally in animal models, and
what obstacles remain before Klotho-based therapies can be tested. In Chapter 13, we detail
what we know about the current potential of the exploration of clinical aspects of Klotho-related tests (like the measurement of serum Klotho levels or the relevance of single-nucleotide polymorphisms (SNPs) in the Klotho gene) in patients with vascular disease. In
Chapter 14, we study whether Klotho SNPs are associated with graft failure in kidney
transplantation recipients, which is a process to which transplant vasculopathy is a contributing factor.
Finally, in Chapter 15, we summarize and discuss the results of this thesis and provide a
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53. K. Lindberg, R. Amin, O. W. Moe, M. C. Hu, R. G. Erben, A. Ostman Wernerson, B. Lanske, H. Olauson, T. E. Larsson, The kidney is the principal organ mediating klotho effects. J. Am. Soc. Nephrol. 25, 2169-2175 (2014). 54. Y. Matsumura, H. Aizawa, T. Shiraki-Iida, R. Nagai, M. Kuro-o, Y. Nabeshima, Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem. Biophys. Res. Commun. 242, 626-630 (1998).
55. N. Koh, T. Fujimori, S. Nishiguchi, A. Tamori, S. Shiomi, T. Nakatani, K. Sugimura, T. Kishimoto, S. Kinoshita, T. Kuroki, Y. Nabeshima, Severely reduced production of klotho in human chronic renal failure kidney. Biochem. Biophys. Res. Commun. 280, 1015-1020 (2001).
56. S. L. Barker, J. Pastor, D. Carranza, H. Quinones, C. Griffith, R. Goetz, M. Mohammadi, J. Ye, J. Zhang, M. C. Hu, M. Kuro-o, O. W. Moe, S. S. Sidhu, The demonstration of alphaKlotho deficiency in human chronic kidney disease with a novel synthetic antibody. Nephrol. Dial. Transplant. 30, 223-233 (2015).
57. J. P. Kooman, M. J. Dekker, L. A. Usvyat, P. Kotanko, F. M. van der Sande, C. G. Schalkwijk, P. G. Shiels, P. Stenvinkel, Inflammation and premature aging in advanced chronic kidney disease. Am. J. Physiol. Renal Physiol. 313, F938-F950 (2017).
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58. H. Kurosu, Y. Ogawa, M. Miyoshi, M. Yamamoto, A. Nandi, K. P. Rosenblatt, M. G. Baum, S. Schiavi, M. C. Hu, O. W. Moe, M. Kuro-o, Regulation of fibroblast growth factor-23 signaling by klotho. J. Biol. Chem. 281, 6120-6123 (2006).
59. I. Urakawa, Y. Yamazaki, T. Shimada, K. Iijima, H. Hasegawa, K. Okawa, T. Fujita, S. Fukumoto, T. Yamashita, Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 444, 770-774 (2006). 60. A. S. Go, G. M. Chertow, D. Fan, C. E. McCulloch, C. Y. Hsu, Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N. Engl. J. Med. 351, 1296-1305 (2004).
61. Y. Saito, T. Yamagishi, T. Nakamura, Y. Ohyama, H. Aizawa, T. Suga, Y. Matsumura, H. Masuda, M. Kurabayashi, M. Kuro-o, Y. Nabeshima, R. Nagai, Klotho protein protects against endothelial dysfunction. Biochem. Biophys. Res. Commun. 248, 324-329 (1998).
62. T. Nakamura, Y. Saito, Y. Ohyama, H. Masuda, H. Sumino, M. Kuro-o, Y. Nabeshima, R. Nagai, M. Kurabayashi, Production of nitric oxide, but not prostacyclin, is reduced in klotho mice. Jpn. J. Pharmacol. 89, 149-156 (2002). 63. D. Gao, Z. Zuo, J. Tian, Q. Ali, Y. Lin, H. Lei, Z. Sun, Activation of SIRT1 Attenuates Klotho Deficiency-Induced Arterial Stiffness and Hypertension by Enhancing AMP-Activated Protein Kinase Activity. Hypertension. 68, 1191-1199 (2016).
64. K. Chen, X. Zhou, Z. Sun, Haplodeficiency of Klotho Gene Causes Arterial Stiffening via Upregulation of Scleraxis Expression and Induction of Autophagy. Hypertension. 66, 1006-1013 (2015).
65. T. Shimada, Y. Takeshita, T. Murohara, K. Sasaki, K. Egami, S. Shintani, Y. Katsuda, H. Ikeda, Y. Nabeshima, T. Imaizumi, Angiogenesis and vasculogenesis are impaired in the precocious-aging klotho mouse. Circulation. 110, 1148-1155 (2004).
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68. Y. Saito, T. Nakamura, Y. Ohyama, T. Suzuki, A. Iida, T. Shiraki-Iida, M. Kuro-o, Y. Nabeshima, M. Kurabayashi, R. Nagai, In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome. Biochem. Biophys. Res. Commun. 276, 767-772 (2000).
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70. K. Lindberg, H. Olauson, R. Amin, A. Ponnusamy, R. Goetz, R. F. Taylor, M. Mohammadi, A. Canfield, K. Kublickiene, T. E. Larsson, Arterial klotho expression and FGF23 effects on vascular calcification and function. PLoS One. 8, e60658 (2013).
71. K. Lim, T. S. Lu, G. Molostvov, C. Lee, F. Lam, D. Zehnder, L. L. Hsiao, Vascular Klotho Deficiency Potentiates the Development of Human Artery Calcification and Mediates Resistance to FGF-23. Circulation. 125, 2243-2255 (2012).
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Chapter 2
The role of the anti-ageing protein Klotho in
vascular physiology and pathophysiology
R. Mencke J.L. Hillebrands on behalf of the NIGRAM Consortium
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Abstract
Klotho is an anti-ageing protein that functions in many pathways that govern ageing, like regulation of phosphate homeostasis, insulin signaling, and Wnt signaling. Klotho expression levels and levels in blood decline during ageing. The vascular phenotype of Klotho deficiency features medial calcification, intima hyperplasia, endothelial dysfunction, arterial stiffening, hypertension, and impaired angiogenesis and vasculogenesis, with characteristics similar to aged human arteries.
Klotho-deficient phenotypes can be prevented and rescued by Klotho gene expression or protein supplementation. High phosphate levels are likely to be directly pathogenic and are a prerequisite for medial calcification, but more important determinants are pathways that regulate cellular senescence, suggesting that deficiency of Klotho renders cells susceptible to phosphate toxicity. Overexpression of Klotho is shown to ameliorate medial calcification, endothelial dysfunction, and hypertension.
Endogenous vascular Klotho expression is a controversial subject and, currently, no compelling evidence exists that supports the existence of vascular membrane-bound Klotho expression, as expressed in kidney. In vitro, Klotho has been shown to decrease oxidative stress and apoptosis in both SMCs and ECs, to reduce SMC calcification, to maintain the contractile SMC phenotype, and to prevent µ-calpain overactivation in ECs.
Klotho has many protective effects with regard to the vasculature and constitutes a very promising therapeutic target. The purpose of this review is to explore the etiology of the vascular phenotype of Klotho deficiency and the therapeutic potential of Klotho in vascular disease.
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Introduction
Klotho is an anti-ageing gene that was discovered in 1997 (1). It is expressed mainly in the kidney, parathyroid gland, and choroid plexus and exists as a membrane-bound protein that can be cleaved off, also to be found as a soluble protein in the blood, urine, and cerebrospinal fluid (2, 3). The Klotho protein contains two homologous internal repeats, termed KL1 and KL2. Soluble Klotho contains both KL1 and KL2, but proteolytic cleavage may also occur between KL1 and KL2, producing two additional, smaller soluble Klotho proteins. An alternatively spliced transcript has also been hypothesized to code for a secreted Klotho protein (4). The current paradigm of Klotho protein forms is summarized in Figure 1.
Deficiency of Klotho in mice was found to have profound systemic effects, producing a phenotype markedly reminiscent of human ageing. This phenotype consists of, among other traits, a short lifespan, stunted growth and kyphosis, vascular calcification and atherosclerosis, osteoporosis, pulmonary emphysema, cognitive impairment, deafness, and atrophy of skin, muscles, gonads, and many other organs (1). It has been shown that increasing Klotho levels in mice yields an extended lifespan (120-130% of normal) (5), better cognitive function (6, 7), resistance against induction of renal disease (8-15), cardiac disease (16, 17), pulmonary disease (18, 19), vascular calcification (3), diabetes (20, 21), oxidative stress (22, 23), while also acting as an in vivo tumor suppressor (9, 24-27).
So far, it has been shown that Klotho acts via at least six distinct mechanisms: (1) as a
membrane-bound co-receptor for soluble ligands (as a co-receptor for ligand fibroblast growth factor (FGF)23 with FGFR1c, inducing phosphaturia, down-regulating the vitamin D-producing enzyme 1α-hydroxylase, and regulating renal sodium re-absorption) (28-32), (2) as
a soluble co-receptor for soluble ligands (maintaining endothelial integrity by mediating vascular endothelial growth factor (VEGF)-induced internalization of the Klotho-bound transient receptor potential cation channel (TRPC)1/VEGFR2 complex (33), (3) as a soluble
decoy receptor for soluble factors (inhibiting Wnt signaling by binding to several Wnt factors) (10, 11, 34, 35), (4) as a soluble protein decreasing receptor affinity for ligands (directly
inhibiting insulin growth factor (IGF)1 and transforming growth factor (TGF)β signaling by binding to IGF1R and TGFβR2, respectively) (5, 9, 24), and (5) as a membrane-bound
competitor for binding sites (inhibiting FGF2 signaling by binding to FGFR1c) (14, 25), and (6)
as an enzyme (modifying sugar moieties on calcium channel TRPV5, potassium channel ROMK1, and phosphate transporter NaPi2a, affecting their cell surface abundance through sialidase or β-glucoronidase activity) (36-40).
Klotho has garnered a lot of attention in vascular biology and a number of observations can be linked to underscore its clinical relevance. The vascular phenotype of Klotho deficiency is very similar to both human ageing and “accelated” ageing observed in chronic kidney disease (CKD) (1). CKD is also a state of acquired Klotho deficiency (3, 41, 42). In humans, genetic
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Klotho deficiency (the H193R missense mutation) also leads to severe vascular calcification (43). It is therefore conceivable that Klotho may play a causal role in the pathogenesis of cardiovascular complications in CKD, the development of which is the leading cause of death in CKD patients (44). Additionally, Klotho gene variants have been found to be protective or detrimental for the development of cardiovascular and cerebrovascular disease (45, 46). Finally, although current serum Klotho measurements may not be reliable, Klotho levels may also be lower in patients with cardiovascular disease (47, 48). It is therefore very important to delineate the effects of Klotho on the cardiovascular system, in order to identify new targets for new therapies. Possible approaches include up-regulation of Klotho, administration of Klotho, or administration of Klotho-based compounds. Structure-function analyses indicate that different domains in the Klotho protein have different functions. FGF23 requires the full-length membrane-bound Klotho protein (31). The KL2 domain is required for binding to TRPC1/VEGFR2 (33), while KL1 can exert tumor suppressor effects and inhibit IGFR and Wnt signaling (26, 34), independently of enzymatic activity, while enzymatic activity in either domain is required for modifying TRPV5 and NaPi2a (37, 39, 49).
Figure 1. Paradigm of Klotho forms. Two Klotho mRNA transcripts are expressed in the kidney, of which one contains a short intronic sequence after exon 3, giving rise to a stop codon. This mRNA transcript has been hypothesized to code for a secreted Klotho protein. The other transcript codes for membrane-bound Klotho (positive immunohistochemical staining on healthy human kidney). Membrane-bound Klotho is cleaved proteolytically by secretases above the membrane and between the KL1 and KL2 internal repeats, producing three soluble Klotho molecules.
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These effects, apparently acting in concert in alleviating ageing from the sub-cellular level to the level to the level of the organism, show that different functions of Klotho can be dissected, which may be of consequence for future therapies. consequence for future therapies. Therefore, this comprehensive review will focus on the role of Klotho in vascular physiology and pathophysiology in order to assess its therapeutic potential. We will first describe the phenotype of Klotho deficiency, the effects of interventions on the phenotype of Klotho deficiency, and the experimental effects of Klotho overexpression, proceeding to the topics of endogenous vascular Klotho expression, and the in vitro effects of Klotho on vascular cells.
The vasculature and Klotho – in vivo experimental evidence
Klotho-hypomorphic kl/kl mice, as originally described, exhibit two remarkable vascular histological features: vascular calcification in the tunica media (the contractile, smooth muscle cell layer of arteries) and intima hyperplasia (hyperplasia of the inner, endothelial lining of arteries, which is invaded by migrating and proliferating smooth muscle cells) (1). Functionally, Klotho deficiency causes endothelial dysfunction and arterial stiffening, as well as hypertension and impaired angiogenesis. We will assess the histological, functional, and molecular phenotype of in vivo Klotho deficiency and its resemblance to the phenotype of the aged human vasculature, as well as assess the effects of interventions in Klotho deficiency (summed up in Tables 1 and 2), assess the effects of Klotho overexpression or supplementation on various vascular phenotypes.
Vascular calcification
Vascular calcification in Klotho deficiency
The vascular calcification in kl/kl mice is progressive from 4 weeks of age onward, mostly confined to the media and reminiscent of Mönckeberg’s sclerosis in human chronic kidney disease (CKD). It is present in arteries ranging from aorta to middle-sized muscular arteries, to small renal arteries. Furthermore, extensive ectopic calcification was noted in brain, lung, gastrointestinal tract, testis, skin, and heart, in line with the overt hypercalcemia, hyperphosphatemia, and hypervitaminosis D (50) found in these mice. These features were noted only in animals homozygous for the hypomorphic kl allele. It has been demonstrated that aorta and kidneys from Klotho-deficienct mice indeed have a significantly higher calcium
26
content (3). Both histochemistry and electron microscopy analyses indicate that these calcifications are largely confined to the elastin fibers of the tunica media (51). Furthermore, the medial smooth muscle cells are phenotypically akin to matrix synthesizing cells surrounded by secreted matrix vesicles. The vascular smooth muscle cells in calcifying areas in kl/kl aorta exhibit high Runx2 expression, indicative of trans-differentiation to an osteoblast-like phenotype (52). Expression of matrix Gla protein (MGP), a potent vitamin K-dependent calcification inhibitor known to be highly expressed in calcifying human arteries, is similarly increased at the edges of the calcified areas. Hu et al. later also described higher Runx2 mRNA expression levels in kl/kl aorta, in addition to higher mRNA levels of phosphate transporters Pit1 and Pit2 and lower mRNA levels of smooth muscle cell (SMC) marker SM22α (3). Higher Pit1 mRNA and Runx2 mRNA and protein levels in kl/kl aorta were also found by other authors, in addition to higher Msx2, osterix, tumor necrosis factor (TNF)-α, alkaline phosphatase, osteopontin, receptor activator of nuclear factor kappa-B ligand (RANKL), nuclear factor of activated T cells (NFAT)5, and Sox9 mRNA and/or protein levels (53-56). These findings suggest that medial calcification in Klotho deficiency is an active process similar to osteogenesis, analogous to medial calcification in human CKD and human ageing. This view is supported by the finding that even expression of otherwise osteocyte-exclusive FGF23 was found to arise in kl/kl aortic calcifications (55). Mammalian target of rapamycin (mTOR) has also been found to be activated in kl/kl mouse aorta, which contributes to medial calcification (57). Furthermore, cyclooxygenase 2, involved in prostaglandin synthesis and bone formation, is also up-regulated in Klotho-/- mice, although at a lower level in aortic calcified lesions as
compared to aortic valve calcifications (58). In addition to MGP and osteopontin up-regulation, the finding of up-regulated stanniocalcin 2 expression (but not stanniocalcin 1 expression) in calcifying lesions in Klotho-deficient aorta and renal arterioles is speculated to indicate a protective mechanism at work as well (59). Kl/kl mouse aorta and kidney may also express higher levels of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and ANK, a pyrophosphate generator and transporter, respectively (60). This possibly reflects activation of an additional protective mechanism, in order to prevent calcium phosphate deposition. Furthermore, it was found that the miRNAs miR-135a*, miR-762, miR-714, and miR-712* were highly up-regulated in kl/kl aorta, which was accompanied by down-regulation of target genes: calcium efflux pumps/exchangers NCKX1, PMCA1, and NCKX4 (an effect found to predispose towards calcification) (61). These authors also note that 10% of (heterozygous) kl/+ animals also display minor vascular calcification, a feature that was associated with up-regulated expression of the aforementioned miRNAs, also in kl/+ animals. No differences have been reported between vascular phenotypes of different Klotho mutant mice, which include the original (hypomorphic) kl//kl mice (1), different Klotho-/- mice (30, 62,
63), and β-actin-Cre/KL-LoxP mice (64). In short, the phenotype of Klotho deficiency features medial calcification, associated with altered expression of both phosphate and calcium transporters, activation of mechanisms that aim to inhibit calcification, and osteochondrogenic transdifferentiation of SMCs. These lesions greatly resemble the calcified
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lesions in human age-related or (exaggeratedly) CKD-related medial calcification, both morphologically and molecularly.
Interventions in the development of vascular calcification
The effect of restoration of Klotho expression in Klotho deficiency
In their original study, Kuro-o et al. describe crossing of kl/kl mice with mice that express Klotho constitutively under the ubiquitous elongation factor 1α promoter, rescuing the phenotype to a large extent (1). Both aortic calcification and ectopic calcification were markedly reduced. The same group then performed an experiment to assess whether the Klotho gene is also able to rescue the developed kl/kl phenotype, rather than prevent it. Using an adenoviral vector containing the Klotho gene via tail vein infusion at 4-5 weeks of age, the development of aortic medial calcification could be halted and was noted, at 27 weeks of age, in the few mice that survived that long, to be less advanced than in 4-10-weeks-old kl/kl mice (65). They then used kl/kl mice that express an ectopic Klotho gene conditionally (under the zinc-dependent mouse metallothionein-I promoter), allowing for free manipulation of Klotho expression. It was also found that there was no development of medial calcification if Klotho expression was induced via zinc water feeding from three weeks of age onwards (66). Furthermore, it was found that inducing Klotho expression for three weeks was already successful in completely reversing medial calcification that had already developed before starting zinc water feeding at 5 or 8 weeks of age, illustrating the potential therapeutic potency of Klotho. It is as of yet unknown how this therapeutic effect can be explained, but a potential to both reverse and prevent age-related disorders (for the phenotypical improvement was systemic) offers tantalizing possibilities. Moreover, after initially preventing development of medial calcification, subsequent zinc withdrawal from 11 weeks of age onwards caused renewed development of medial calcification (assessed at 19 and 27 weeks of age). This illustrates that Klotho is continuously required in order to maintain vascular health, rather than only during a hypothetical critical period after birth. It also underscores how acquired Klotho deficiency later in life, as occurs in CKD, may as a single factor be enough to materialize a phenotype of vascular calcification.
Restoration of Klotho expression in Klotho deficiency affects the phenotype via soluble Klotho While it is evident from the previous discussion that genetic re-introduction of Klotho can rescue the vascular calcification in Klotho deficiency, it is not immediately clear whether these effects are mediated by membrane-bound or soluble Klotho. It could be argued that soluble Klotho is the likely mediator, since the induced non-vascular expression patterns differ greatly. Induced Klotho expression is mostly confined to the gastrointestinal tract in the conditional zinc-dependent model (66), confined to the liver in the adenovirus-mediated
28
model (65), and found in many organs in the ubiquitous overexpression model (1), but all models produce a similar effect. There is, however, more compelling evidence that shows that the rescue of the vascular kl/kl phenotype is at least predominantly dependent on soluble Klotho. First of all, a study by Chen et al. shows that intraperitoneal injections of soluble Klotho (0.02 mg/kg/48 h between 3 and 8 weeks of age) result in marked reduction of vascular calcification (67). Notably, while increasing urinary phosphate excretion, this treatment left serum phosphate and calcium levels unaltered. Secondly, a number of studies by a group that has created a number of organ-specific Klotho knockout models has yielded important insights. Lindberg et al. showed that the vascular kl/kl phenotype is present in Six2-Cre/KL-LoxP mice, a mouse model of selective and complete renal tubular Klotho deficiency, suggesting that vascular calcification is normally prevented by kidney-derived Klotho (68). In further support of this conclusion, this study also shows that the soluble Klotho levels in the blood are largely kidney-derived. Distal tubule Klotho deletion in Ksp-Cre/KL-LoxP mice, however, did not induce vascular calcification (64), suggesting that moderate remaining Klotho levels are enough to prevent the overt phenotype, akin to the lack of vascular calcification in kl/+ mice. Selective deletion of Klotho in arterial smooth muscle cells (in SM22a-Cre/KL-LoxP mice) did not produce vascular abnormalities (69). This suggests that if any endogenous expression of Klotho in smooth muscle cells is present, its deletion alone does not contribute significantly to the development of an overtly aberrant phenotype. Moreover, neither deletion of parathyroid Klotho in PTH-Cre/KL-LoxP mice, nor deletion of proximal tubule Klotho in Kap-Cre/KL-LoxP mice, PEPCK-Cre/KL-LoxP mice, or Scl34a1-Cre/KL-LoxP mice induced an overt vascular phenotype (70, 71). A final important argument for soluble Klotho-mediated vasculoprotection is derived from the parabiosis experiment by Saito et al., showing that a shared circulatory system with WT mice restores acetylcholine-dependent vasodilation in kl/+ mice after 4 weeks (72).
Interventions in mineral homeostasis
The first interventions aimed at rescuing the kl/kl phenotype involved interventions in mineral metabolism. An early study shows that reducing dietary phosphate (0.4% vs 1.03%) in kl/kl mice partially rescues many features of their phenotype, among which reduction of ectopic calcification in kidney (in male mice, and in female mice as well after addition of 0.25% zinc orotate) (73). Vascular calcification was not examined, but it is likely that vascular calcifications were reduced as well. These effects, however, may have been dependent at least in part on a phosphate restriction-induced increase in renal Klotho expression, even in Klotho-hypomorphic kl/kl mice, which are not fully deficient.
Subsequent studies have yielded extensive evidence indicative of a sine qua non role for phosphate toxicity in vascular calcification in complete Klotho deficiency. Lowering phosphate levels through a genetic approach (in NaPi2a-/-/Klotho-/- mice) was shown to prevent vascular
29
re-established the phenotype of vascular calcification (75). These results indicate that high phosphate levels are a requirement (in the setting of Klotho deficiency) for the vascular calcification to develop despite the presence of hypercalcemia and extremely elevated 1,25(OH)2-vitamin D3 levels. NaPi2a expression was found to be increased in kl/kl kidney,
which is thought to cause hyperphosphatemia through increased phosphate re-absorption, a hypothesis that is supported by the lack of hyperphosphatemia in the NaPi2a-/-/Klotho-/- mice.
It is unknown whether other vascular features of Klotho deficiency may also be rescued by normalization of phosphate levels. Curiously, in another experiment, a low phosphate diet (0.2%) in full Klotho-/- mice was unable to rescue renal calcification after 7 weeks (assuming
that vascular calcifications were also still present) (30). The low phosphate diet, however, only caused a decrease in serum phosphate level of 0.7 mg/dL (whereas a 3.6 mg/dL decrease was reported in the genetic study (75) at 6 weeks of age, although the actual phosphate levels and assays may not be comparable). It is therefore possible that despite the low phosphate diet, the serum phosphate levels remained elevated to the point of causing calcifications. This may be mediated via increased NaPi2a activity and without the possibility of compensatory Klotho up-regulation, as was shown to be possible in Klotho-hypomorphic kl/kl mice (73). Interestingly, a low calcium diet (0.02%) was shown to be effective in preventing ectopic calcifications in this study, showing that calcium can also function as a rate-limiting factor in the pathophysiology of Klotho-/- vascular calcification, in addition to phosphate (30). An
attempt to target phosphate homeostasis by ablating secreted frizzled-related protein 4 (Sfrp4) in Sfrp4-/-/Klotho-/- mice did not improve or worsen vascular calcification (76). Since
Sfrp4-/- mice display no mineral homeostasis abnormalities, these data suggest that Sfrp4
does not play a significant role in phosphate regulation. Finally, deletion of both Klotho and dentin matrix protein 1 (DMP1) underlined the importance of DMP1 in phosphate homeostasis(77). Klotho-/-/DMP1-/- mice displayed more severe vascular calcification and
more apoptosis in aorta and arterioles than did Klotho-/- mice, at similar serum phosphate
levels. If DMP1 expression is increased in Klotho-/- arteries as it is in the kidney, this may
signify activation of another local mechanism that protects against Klotho deficiency-induced calcification. Hypophosphatemia in DMP-/- mice is converted to hyperphosphatemia upon
additional knockout of Klotho, which improves bone mineralization, but the lack of DMP1 apparently leaves arteries particularly vulnerable to calcification.
Another approach that has been studied is the modulation of vitamin D levels that have been reported to be extremely elevated in Klotho deficiency (50). Dietary vitamin D restriction was also shown to be effective in reducing vascular calcification in complete Klotho-/- mice (30). In
the vitamin D-deficient diet that was used, however, phosphate content was also decreased from 1.09% to 0.4% and calcium content was decreased from 1.46% to 0.6%. This offers other possible explanations for the phenotypic amelioration, perhaps to be attributed to synergistic lower phosphate, calcium, and vitamin D levels. It was noted that in control experiments with diets with the same phosphate and calcium content, rescue of the phenotype was not observed. However, it is difficult to account for the interactions between these mediators of
30
mineral homeostasis, e.g. for the additional effect of vitamin D deficiency on phosphate levels, which may result in a difference in phenotype. Using a genetic approach, ablation of 1α-hydroxylase expression in Cyp27b1-/-/Klotho-/- mice completely prevented the
development of vascular and ectopic calcifications (78). However, this genetic intervention also caused hypophosphatemia and hypocalcemia, suggesting that vitamin D effects on the vasculature are mediated, at least in part, by phosphate and calcium. These findings concerning serum biochemistry and renal calcifications have been corroborated in another study (79). In a similar study, it was shown that ablation of vitamin D signaling, by mutation of the vitamin D receptor in VDRΔ/Δ/Klotho-/- mice also prevents vascular calcification at least
at 8 weeks of age (80). It should be noted that these mice were on a rescue diet enriched in phosphate (1.25%), calcium (2.0%), lactose (20%), and 600 IU vitamin D/kg, ensuring normocalcemia, normophosphatemia, and normal PTH levels, so again, in this experiment, rescue of the phenotype might be confounded by normalized phosphate and calcium levels. A study by Alexander et al. compared Klotho-/- mice on a control diet (0.9% calcium, 0.63%
phosphate, 1500 IU 1,25(OH)2-vitamin D3) to Klotho-/- mice on a rescue diet (0.34% calcium,
0.22% phosphate, <5 IU 1,25(OH)2-vitamin D3) (40). In this study, while serum vitamin D and
calcium levels normalized, phosphate levels were still elevated and the development of renal calcifications was only halted slightly. Vascular calcification was not assessed.
Moving up a step in regulatory mechanisms to phosphaturic hormones, the identical phenotypes with regard to mineral metabolism in FGF23-/- mice, Klotho-/- mice, and FGF23
-/-/Klotho-/- mice constitute compelling evidence for FGF23 and Klotho acting in a common
pathway (63). FGF23 action was determined to be dependent on Klotho due to the inability of FGF23 to induce phosphaturia in Klotho-/- mice, of which it is capable in wild-type and
FGF23-/- mice. In a similar study, it was found that Hyp/Klotho-/- mice (harboring mutations in
the Klotho and PHEX genes, the latter of which causes high FGF23 levels and hypophosphatemia) basically exhibit a Klotho-/- phenotype with vascular calcification and
hyperphosphatemia(60). This supports the notion that FGF23 regulation of phosphate is fully Klotho-dependent. Finally, the finding that Klotho-/-/FGF23TG mice (lacking Klotho and
overexpressing FGF23) also display vascular calcification similar to Klotho-/- mice
independently confirms this line of evidence of FGF23 signaling effects on mineral metabolism being Klotho-dependent (81). Another group has generated PTH-/-/Klotho-/- mice, in which
ectopic calcifications are still pervasive, in the presence of even more exaggerated hyperphosphatemia and normocalcemia (yielding a Ca x P product similar to Klotho-/- mice)
(82). Although the vasculature was not investigated, it is reasonable to hypothesize that vascular calcifications were also still present. Following the same train of thought, vascular calcified lesions may be alleviated in PTH-infused Klotho-/- mice that were shown to exhibit
normophosphatemia (82, 83). These results suggest that, although there are many interactions between PTH and Klotho, they influence vascular calcification largely independently via regulating phosphate and calcium levels.
31
The previous discussion favors a direct pathogenic role for phosphate and calcium, whereas vitamin D effects are most likely indirect and mediated by phosphate and calcium. However, a lot of evidence is still circumstantial and an experiment in which vitamin D signaling ablation is combined with a high phosphate diet as compared to both interventions alone might provide clearer answers as to whether vitamin D effects on calcification are exclusively mediated by its target electrolytes.
Interventions in osteochondrogenic signaling
In a study exploring the effects of treatment with the aldosterone receptor antagonis spironolactone on kl/kl mice, it was found that aortic calcification was markedly diminished, potentially owing to less osteoinductive signaling due to lower Pit1 levels (53). Levels of Runx2, Msx2, osterix, TNF-α, and alkaline phosphatase mRNA were also lowered by spironolactone treatment. Interestingly, plasminogen activator inhibitor (PAI)-1 mRNA expression was reduced by spironolactone and endothelial nitric oxide synthase (eNOS) mRNA expression was normalized, suggesting that spironolactone may also have improved endothelial function, although this was not assessed in this study. The role of eNOS itself was recently addressed when it was found that treatment of kl/kl mice with homoarginine exacerbated vascular calcification (56). Homoarginine treatment essentially resulted in NOS inhibition and subsequent osteoinductive signaling, as determined by increased levels of Cbfa1, Pai1, Msx2, and alkaline phosphatase mRNA (however, coupled with attenuation of apoptosis). As will be discussed in section 2.3.1, Klotho deficiency entails impaired NO production, but the homoarginine-induced aggravation of vascular calcification indicates that the residual NO production still offers some degree of protection against vascular calcification. In a recent study, it was found that 0.28 M NH4Cl in drinking water greatly
reduced vascular calcification in kl/kl mice, whereas vitamin D, calcium, and phosphate levels were unaltered (54). Acidosis, which is already a feature of Klotho deficiency, was slightly aggravated by NH4Cl treatment, but the difference did not reach significance. A lower blood
pH may have negatively affected calcium and phosphate precipitation slightly, but lysosomal alkalinization and subsequent osmosensitive NFAT5 down-regulation were likely to be a greater contributor. This prevented downstream Runx2-mediated osteochondrogenic signaling in smooth muscle cells and normalized senescence-associated TGFβ, PAI-1, p21, and senescence-associated (SA)-β-galactosidase mRNA levels. Aiming to address to which property of NH4Cl these effects can be attributed, Leibrock et al. have also tested NH4NO3 (a
different NH4+ donor) (84), acetazolamide (which induces acidosis) (85), and NaHCO3 (which
induces alkalosis) (86). Treatment with 0.28 M NH4NO3 until 8 weeks of age prevented the
development of vascular calcification as well, without affecting serum phosphate of calcium levels (84). Acetazolamide-induced aggravation of acidosis was also found to prevent medial calcification in kl/kl mice, an effect indeed likely mediated by increased solubility of osteoinductive calcium phosphate crystals, as well as by a decrease in aldosterone levels. The
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inhibition of osteochondrogenic signaling was also exemplified by normalization of Klotho deficiency-induced elevations of aortic alkaline phosphatase and of calcification inhibitors osteopontin, osteoprotegerin, and fetuin A (85). Illustrating the effects of alkalosis, however, treatment with 0.15 M NaHCO3 also partially reversed ectopic calcification (arteries were not
investigated, but may also have been less affected). This effect was likely also the result of lower aldosterone levels, as well as the result of alkalosis-induced phosphaturia, concordant with lower serum phosphate (but not calcium) levels (86). Aiming to modulate active calcification by targeting cyclooxygenase 2, it was found that both a genetic approach (in Ptgs2+/-/Klotho-/- and Ptgs2-/-/Klotho-/-mice) and a pharmacological approach (using celecoxib
in Klotho-/- mice) resulted in less calcification of the aortic valve (58). Celecoxib treatment
inhibited osteochondrogenic signaling, as evidenced by decreased Runx2, osteopontin, and alkaline phosphatase mRNA levels. Aortic calcification, however, was not analyzed in the genetic model and was similar in Klotho-/- mice fed a normal or a celecoxib diet, a result that
seems in line with lower aortic cyclooxygenase 2 expression in these animals. Finally, the same group also found that aortic valve calcification in Klotho-/- mice was dependent on bone
morphogenetic protein (BMP) signaling via pSmad1/5/8 in aortic valce interstitial cells (87). However, pSmad1/5/8 was not detected in aortic SMCs, so the relevance of BMP signaling to vascular calcification in Klotho deficiency is yet to be determined.
Interventions in senescence-related pathways
The relevance of the decrease in the expression of PAI-1, a known inducer of cellular senescence, is especially evident in a study on PAI+/-/kl/kl and PAI-/-/kl/kl mice (88). Renal
calcifications were reduced by 41% and 96%, respectively (aortic calcification was not assessed), while mice displayed comparable hyperphosphatemia and hypercalcemia, as compared to kl/kl mice. Klotho deficiency-induced up-regulation of a down-stream target of PAI-1, p16Ink4a, a known tumor suppressor that induces cellular senescence, was found to be
decreased by partial and full PAI-1 deletion. A recent study uncovered an interesting additional link between PAI-1, p16Ink4a, and Klotho, when it was found that the Klotho
deficiency phenotype including ectopic calcification was partially rescued in p16Ink4a-/-/kl/kl
mice. This was due to de-repression of p16Ink4a-induced E2F1- and E2F3-mediated
down-regulation of residual Klotho expression (89). This is in line with p16Ink4a-/-/Klotho-/- mice being
phenotypically identical to Klotho-/- mice, although the vascular phenotype in these mice was
not specifically disclosed. The study by Eren et al. demonstrates that, although high phosphate and calcium levels are probably required for the development of calcifications, other factors are also capable of influencing the phenotype. Apparently, modulating cellular susceptibility to noxious stimuli may impede the development of vascular calcification, without directly altering phosphate and calcium levels. Another argument for this less phosphate-centric view is a study in which a central role was proposed for µ-calpain overactivation in the development of kl/kl phenotypes (55). Their data show that µ-calpain
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inhibitor BDA-410, administered intraperitoneally at a dose of 100 mg/kg/day between 2 and 6 weeks of age, completely prevented the development of aortic calcification and associated arterial wall thickening at 6 weeks of age. Hyperphosphatemia, hypercalcemia, and hypervitaminosis D were comparable to kl/kl levels. This was apparently also the case if mice were treated between 4 and 6 weeks of age. Aortic mRNA levels of FGF23, Runx2, osteopontin, and RANKL were reduced after BDA-410 treatment, suggesting prevention of osteochondrogenic differentiation of smooth muscle cells. Cortical calcifications in the kidney, however, were still present to a minor extent and were hypothesized to develop due to dysregulated calcium re-absorption, rather than due to a process similar to vascular calcification.
Interestingly, rapamycin-induced inhibition of mTOR signaling (1.2 mg/kg/day between 3 and 4 weeks of age), while blunting induction of Cbfa1 expression, greatly ameliorated vascular calcification in CKD mice, but not at all in Klotho-/- mice (or ex vivo in Klotho-/- aorta rings) (57).
This cements Klotho as a key downstream mediator of rapamycin. Targeting a different pathway, it was reported that partial ablation of insulin receptor substrate (IRS) in IRS
+/-/Klotho-/- mice also prevents the development of vascular calcification, probably due to
inhibition of IGF1- and insulin-induced senescence. The previous discussion endorses the view that dysregulation of mineral homeostasis in Klotho deficiency is only part of the pathogenesis and that Klotho has a profound effect on pathways that regulate apoptosis and cellular senescence. Deficiency of Klotho may render cells susceptible to phosphate toxicity. The emergent paradigm for contributors to Klotho deficiency-induced vascular calcification is depicted in Figure 2.
Miscellaneous interventions
It was noted that leptin-deficient ob/ob/Klotho-/- mice still displayed vascular calcification, but
it is unclear how the phenotype compares to Klotho-/- mice (90). This would be interesting to
address since it could be hypothesized that vascular lesions may be aggravated due to leptin deficiency in addition to Klotho deficiency. High-fat diet-induced vascular calcification in kl/+ mice was ameliorated by AMP-activated protein kinase activator AICAR (91). Treatment of Klotho-/- mice with rikkunshito, aiming to increase ghrelin signaling, did not result in an effect
on vascular calcification(92), suggesting that ghrelin signaling may not be of relevance in vascular calcification in Klotho deficiency.
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Effects of Klotho overexpression/supplementation on vascular calcification
A study by Hu et al., using the transgenic KL-Tg mice originally described by Kuro-o et al., found that Klotho overexpression causes mice with CKD (induced by nephrectomy + ischemia/reperfusion injury) to display very little or no vascular calcification and lower aortic calcium content than WT CKD mice (3). There are multiple possible mechanistic explanations for this. Firstly, these mice exhibit less severe vascular calcification, because CKD in these mice is relatively mild and creatinine levels do not rise significantly. Secondly, the lack of increase in phosphate serum level and fractional excretion due to phosphaturic actions of Klotho may also render these mice less prone to developing vascular calcification. Thirdly, the higher serum Klotho levels may have a direct protective effect on the vasculature. Corrected for serum phosphate and creatinine, however, the KL-Tg mice still have the lowest calcium content, so there is an additional vasculoprotective effect mediated by Klotho. Overexpression of Klotho down-regulated Pit1, Pit2, and Runx2, while up-regulating SM22α in KL-Tg aorta as compared to WT controls, possibly reflecting Klotho-driven smooth muscle cell differentiation towards a contractile phenotype. Using a
Figure 2. Paradigm of contributing factors in Klotho deficiency-induced vascular calcification. Vitamin D causes high calcium and phosphate levels, which contribute to the induction of vascular calcification, in smooth muscle cells that have undergone osteochondrogenic transition and are senescent due to plasminogen activator inhibitor (PAI)-1 overexpression, µ-calpain overactivation, and increased insulin signaling. Anti-calcification mechanisms are activated, but are overwhelmed by the effects of Klotho deficiency.
