University of Groningen

Klotho in vascular biologyMencke, Rik

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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

Published in: Ageing Res Rev. 2017 May;35:124-146

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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

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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

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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 calcification at least until 12 weeks of age (74). A high phosphate diet (1.2%) in the same mice

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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

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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.

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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.

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Table 1. Effects of interventions on vascular calcification in models of Klotho deficiency

Intervention Animal model Effect on phenotype Reference Constitutive Klotho gene expression

EF1α-KL/kl/kl mice Prevention of VC Kuro-o, 1997

Re-introduction of Klotho gene expression

Ad-KL Kl/kl mice Rescue of VC Shiraki-Iida, 2000

Early induction of Klotho gene expression

MTm-KL Kl/kl mice Prevention of VC Masuda, 2005

Late induction of Klotho gene expression

MTm-KL Kl/kl mice Rescue of VC Masuda, 2005

Cessation of induction of Klotho gene expression

MTm-KL Kl/kl mice Re-development of VC

Masuda, 2005

Klotho protein i.p. Kl/kl mice + Klotho i.p. Reduction of VC Chen, 2012 Whole nephron Klotho deletion Six2-Cre/KL-LoxP mice Development of VC Lindberg, 2014 Distal tubule Klotho deletion Ksp-Cre/KL-LoxP mice Prevention of VC Olauson, 2013 Proximal tubule Klotho deletion Kap-Cre/KL-LoxP mice Prevention of VC Ide, 2016 Proximal tubule Klotho deletion PEPCK-Cre/KL-LoxP mice Prevention of VC Ide, 2016 Proximal tubule Klotho deletion SLC4a1-Cre/KL-Loxp mice Prevention of VC Ide, 2016 Arterial Klotho deletion SM22a-Cre/KL-LoxP mice Prevention of VC Lindberg, 2013 Parathyroid Klotho deletion PTH-Cre/KL-LoxP mice Prevention of VC Olauson, 2013 Dietary phosphate restriction Kl/kl mice + low phosphate

diet Reduction of EC Morishita, 2001

Genetic phosphate reduction NaPi2a-/-/Klotho-/- mice Prevention of VC Ohnishi, 2009 Genetic phosphate reduction NaPi2a-/-/Klotho-/- mice Prevention of VC Ohnishi, 2010 Genetic phosphate reduction + dietary phosphate overload

NaPi2a-/-/Klotho-/- mice + high phosphate diet

Development of VC Ohnishi, 2010

Dietary phosphate restriction Klotho-/- mice + low phosphate diet

Development of EC Tsujikawa, 2003

Dietary calcium restriction Klotho-/- mice + low calcium diet

Prevention of EC Tsujikawa, 2003

Deletion of Sfrp4 Sfrp4-/-/Klotho-/- mice Development of VC Christov, 2011 Deletion of DMP1 DMP1-/-/Klotho-/- mice Exacerbation of VC Rangiani, 2012 Dietary vitamin D restriction Klotho-/- mice + low vitamin

D diet Reduction of VC Tsujikawa, 2003

Genetic vitamin D reduction Cyp27b1-/-/Klotho-/- mice Prevention of VC Ohnishi, 2009 Genetic vitamin D reduction Cyp27b1-/-/Klotho-/- mice Prevention of EC Woudenberg-

Vrenken, 2012 Genetic ablation of vitamin D signaling

VDRΔ/Δ/Klotho-/- mice Prevention of VC Anour, 2012

Dietary calcium, phosphate, and vitamin D restriction

Klotho-/- mice + low Ca, P, vitamin D diet

Reduction of EC Alexander, 2009

Deletion of FGF23 FGF23-/-/Klotho-/- mice Development of VC Nakatani, 2009 Supplementation of FGF23 Klotho-/- mice + FGF23

injection Unaltered hyperphosphatemia

Nakatani, 2009

Deletion of PHEX Hyp/Klotho-/- mice Development of VC Nakatani, 2009 Overexpression of FGF23 Klotho-/-/FGF23TG mice Development of VC Bai, 2008

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Deletion of PTH PTH-/-/Klotho-/- mice Development of EC Yuan, 2012 Suppletion of PTH Klotho-/- mice + PTH infusion Normophosphatemia Yuan, 2011 Inhibition of aldosterone signaling Klotho-/- mice +

spironolactone Reduction of VC Voelkl, 2013

Homoarginine treatment Kl/kl mice + 20 mg/L homoarginine in drinking water

Exacerbation of VC Alesutan, 2016

Lysosomal alkalinization Kl/kl mice + 0.28 M NH4Cl Prevention of VC Leibrock, 2015 Lysosomal alkalinization Kl/kl mice + 0.28 M NH4NO3 Reduction of VC Leibrock, 2016 Induction of acidosis Kl/kl mice + acetazolamide Prevention of VC Leibrock, 2015

Induction of alkalosis Kl/kl mice + 0.15 M NaHCO3 Reduction of EC Leibrock, 2015 Partial cyclooxygenase 2 deletion Ptgs2+/-/Klotho-/- mice Reduction of AVC Wirrig, 2015 Full cyclooxygenase 2 deletion Ptgs2-/-/Klotho-/- mice Reduction of AVC Wirrig, 2015 Cyclooxygenase 2 inhibition Klotho-/- mice + celecoxib Reduction of AVC,

development of VC Wirrig, 2015

Partial deletion of PAI-1 PAI+/-/kl/kl mice Reduction of EC Eren, 2014 Full deletion of PAI-1 PAI-/-/kl/kl mice Prevention of EC Eren, 2014 Deletion of p16Ink4a p16Ink4a-/-/kl/kl mice Prevention of EC Sato, 2015 Deletion of p16Ink4a p16Ink4a-/-/Klotho-/- mice Development of EC Sato, 2015 Inhibition of µ-calpain Klotho-/- mice + BDA-410 i.p. Prevention of VC Nabeshima,

2014 Inhibition of mTOR signaling Klotho-/- mice + rapamycin Development of VC Zhao, 2015 Partial ablation of insulin signaling IRS+/-/Klotho-/- mice Prevention of VC Kurosu, 2005 Deletion of leptin Ob/ob/Klotho-/- mice Development of VC Ohnishi, 2011 AMPK activation Kl/+ mice + high-fat diet Prevention of VC Lin, 2016 Activation of ghrelin signaling Klotho-/- mice + 1000

mg/kg/day (p.o.) rikkunshito Development of VC Fujitsuka, 2016

VC, vascular calcification; EC, ectopic calcification; AVC, aortic valve calcification

pharmacological rather than a genetic approach, concurrently elevated Klotho expression levels induced by rapamycin, vitamin D receptor agonists and the calcitonin gene-related peptide family member intermedin1-53 have also been found to improve vascular calcification in WT mice. This suggests that the beneficial effects of these compounds are partially Klotho-dependent (57, 93, 94).

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Intima hyperplasia

Intima hyperplasia in Klotho deficiency

The other important arterial feature of Klotho deficiency is intima hyperplasia (usually the result of de-differentiation of smooth muscle cells, followed by proliferation and migration to the intima). It was described to be present only in middle-sized muscular arteries (1), whereas one group notes hyalinous sclerosis of interlobar arteries in 6-week-old kl/kl mice (95). These hallmarks have gained virtually no attention in later studies and their characteristics have therefore not been reported.

Klotho overexpression and intima hyperplasia

Experiments aiming to influence the development of intima hyperplasia in Klotho deficiency have not been published to date, but Klotho overexpression and KL1 supplementation have been employed in other models. Data from a study in streptozotocin-injected rats on a high-fat diet seem to indicate that Klotho gene delivery prevents intima hyperplasia in this model (96). Furthermore, a recent study, examining the effect of soluble Klotho (0.01 mg/kg, 5 days/week, between 12 and 20 weeks of age) on the development of atherosclerosis in ApoE-

/- mice, found no effect of Klotho on atherosclerotic lesion area (97). This may be due to the timing of the intervention with respect to plaque formation or a difference in pathogenesis of atherosclerosis between the ApoE-/- model and other models, but blood pressure was also unexpectedly unaltered in these mice. On the whole, Klotho effects on intima hyperplasia are still to be investigated more elaborately.

Endothelial dysfunction

Endothelial dysfunction in Klotho deficiency

Klotho deficiency in mice is known to cause overactivation of µ-calpain by 2 weeks of age, leading to depletion of endogenous calpain inhibitor calpastatin by 3 weeks of age (at which point calcifications have not yet developed). Ultimately, this causes cleavage of calpain substrate αII-spectrin by 4 weeks of age, which would predispose cells to cell death (98). Akin to molecular changes in human aged arteries, aortas of kl/kl mice were also found to exhibit more calpain and capase 3 activity in zymography assays and calpain substrate αII-spectrin cleavage was shown to be increased significantly. Furthermore, in kl/kl aorta, a higher Bax/Bcl2 mRNA ratio was found and both endothelial cells (ECs) and SMCs exhibit an

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increased rate of apoptosis (33, 56). Endothelial apoptosis leads to compromised integrity of the endothelium and it was shown that the kl/kl mouse endothelium is hyperpermeable, as assessed using Evans Blue dye in vivo, already in mice that were only 2 weeks old.

Compromise of endothelial integrity leads to endothelial dysfunction, which is another hallmark of human arterial ageing and leads to intima hyperplasia. This was also demonstrated in Klotho deficiency as impaired vasodilation upon acetylcholine stimulation in aortas (kl/+ mice) and arterioles (kl/kl and kl/+ mice) (72, 99). Aortas of kl/kl mice were too severely calcified to dilate in response to acetylcholine. Also noted were decreased sensitivity (higher ED50) to acetylcholine and lower urinary excretion of NO2- and NO3- in kl/+ mice, a finding that was later also reported in kl/kl mice (100) and that is unsurprising, given the lower bioavailability of NO in aging. There was no difference in arterial dilatation or ED50 upon stimulation with sodium nitroprusside, indicating that the impaired vasodilation in Klotho deficiency is endothelium-dependent and at least in part due to impaired endogenous NO production. Further evidence for this conclusion is the lack of a difference in urinary NO2- and NO3- after NOS inhibitor (L-NAME) treatment. In a follow-up study, these findings were replicated and expanded upon: after administration of L-NAME, differential Klotho allele status caused no difference in relaxation. This is in line with the notion of Klotho deficiency-induced impairment of endothelial NO production as evidenced by decreased relaxation of kl/+ aortas upon stimulation with lecithinized superoxide dismutase (which protects NO from degradation). In addition, reduced NO synthase and VEGF protein expression levels are found in kl/+ and kl/kl aorta (101). Lower eNOS and higher levels of senescence-associated genes PAI-1, TGF-β, p21, and SA-β-galactosidase were noted to be up-regulated in kl/kl aorta (53, 54). Increased PAI-1 mRNA expression and increased PAI-1 protein levels were also described in both smooth muscle and endothelial cells (and kidney) (95). Increased PAI-1 expression in Klotho deficiency was found to be associated with senescence and increased microvascular thrombosis in kidney, and telomere shortening in aorta, as reported by others (88). The presence of increased PAI-1 serum levels and PAI-1 expression in sclerotic vascular lesions in Klotho deficiency, as in aging human arteries, is hypothesized to contribute to thrombosis and extracellular matrix protein accumulation by inhibition of proteolysis. In short, the phenotype of Klotho deficiency also features endothelial dysfunction due to impaired NO synthesis, increased PAI-1 expression, and vascular hyperpermeability due to increased µ-calpain activity. In terms of vascular senescence and deterioration of endothelial function, the molecular aspects of Klotho deficiency appear to be a valid model for human arterial ageing.

Interventions in the development of endothelial dysfunction

The parabiosis experiment described by Saito et al. is the only experiment in which endothelial function was used as the outcome measure of an intervention in Klotho deficiency (72). In this experiment, it was shown that when the circulatory systems of kl/+ mice and WT mice are connected for 4 weeks, acetylcholine-dependent vasodilation in kl/+ mice is

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restored. This indicates that soluble factors, differentially present in Klotho deficiency, are capable of rescuing endothelial dysfunction in this model and circulating Klotho itself would be a prime candidate for this hypothesis.

Klotho overexpression and endothelial dysfunction

An early study explored the effects of Klotho gene transfer in OLETF rats, which significantly improved endothelial function after three weeks, as evidenced by an increase in acetylcholine-induced relaxation (62% compared to 82%) (102). This difference was abolished by L-NAME treatment, suggesting that it was due to a difference in endogenous NO production. Indeed, Klotho gene delivery significantly increased the urinary excretion of NO metabolites. Furthermore, systolic blood pressure and coronary perivascular fibrosis and medial hypertrophy were reduced in Klotho-transfected animals. Another group found that 7 days after gene delivery, Klotho leads to an increase in manganese superoxide dismutase (Mn-SOD) protein levels and activity in mice and in spontaneously hypertensive rats (SHR) (103). This effect was abolished in rats by L-NAME treatment, while Klotho also increased NO metabolite production and decreased lipid peroxide production in mice. However, systolic blood pressure in SHR was not affected by Klotho overexpression, possibly due to the short study period. Taken together, Klotho appears to be a promising candidate for the prevention of endothelial dysfunction.

Arterial stiffening

Arterial stiffening in Klotho deficiency

Not wholly unrelated to endothelial dysfunction and hypertension, kl/+ mice develop arterial stiffening, as is evident from the higher arterial pulse wave velocity at least from 14 weeks of age onwards (104). It is likely that arterial stiffening has not yet fully developed at 6 to 9 weeks of age, at which point kl/+ mice are known to still have a normal sodium nitroprusside response (but already an increased norepinephrine-induced contraction in kl/+ aorta, which was abolished by L-NAME treatment) (101). Histologically, the aorta (but not smaller arteries) was found to contain more collagen-I and less elastin, as well as higher matrix metalloproteinase (MMP)-2, MMP-9, and TGF-β1 expression, all features very similar to the development of age-related arterial stiffening in humans (91, 104). Moreover, Klotho deficiency was associated with increased expression of aldosterone-induced collagen transcription factor scleraxis, with autophagy activation (increased LC3-II/LC3-I ratio), and with the presence of more aortic α-SMA-positive cells. These findings, combined with the development of arterial stiffening preceding the development of hypertension, were taken to

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mean that arterial stiffening was a primary, rather than secondary process. Since the kl/+ mice do not exhibit such severe spontaneous vascular calcifications as found in the full knockout, kl/+ mice may be a suitable model for normal, mild, human ageing, apparently also exhibiting arterial stiffening, which is a major hallmark of human ageing.

Interventions in the development of arterial stiffening

Arterial stiffening in kl/+ mice was found to be improved by eplerenone treatment, which also prevented increased collagen-I deposition, elastin degradation, MMP-2 and MMP-9 expression, and myofibroblast differentiation (104). Arterial stiffening in kl/+ mice was found to develop in as little as 5 weeks on a high-fat diet (HFD) (91). Up-regulation of aortic collagen-I, Runx2, and TGFβ1 levels (but not of tropoelastin levels) was also exacerbated by HFD, while phosphorylated AMP-activated protein kinase (pAMPKα), p-eNOS, and Mn-SOD levels were decreased. Activation of cAMP-dependent AMPK by AMP analogue AICAR reversed the HFD-induced incease in pulse wave velocity, as well as the baseline increase in pulse wave velocity. Phosphorylation of eNOS, urinary nitrate/nitrite, and the expression level of Mn-SOD were also increased by AICAR treatment, and superoxide, collagen-I, Runx2, TGFβ1 levels, and number of elastin breaks were decreased in aorta, without change in tropoelastin levels. It would be very interesting to assess the effect of Klotho overexpression on arterial stiffening, which has not been published to date.

Hypertension

Blood pressure in Klotho deficiency

It was found by Zhou et al. that kl/+ mice develop hypertension from 15 weeks of age onward, as well as increased salt sensitivity, as shown by an additional increase in systolic blood pressure by a 2% saline diet (105). Chen et al. corroborated the increase in blood pressure in kl/+ mice from 16 weeks of age onward, which was preceded by arterial stiffening (104). In another study, the same group re-affirmed the development of hypertension and linked it to higher aldosterone levels, with coincident higher adrenal CYP11B2 expression in kl/+ mice (106). Kl/kl mice on a normal diet were shown to have a lower blood pressure than kl/kl mice after 4 weeks of having been on a low vitamin D, which in turn had a lower blood pressure than WT mice (107). Klotho-/-/VDRΔ/Δ mice on a vitamin D/phosphate/calcium rescue diet were also shown to have a lower mean arterial pressure and blood volume than WT or VDRΔ/Δ mice (32). These seemingly contradictory findings can be explained by the Klotho-dependent effect of FGF23 to stimulate Na+/Cl--co-transporter (NCC)-mediated sodium re-absorption in the distal convoluted tubule. This effect would act in concert with the previously reported

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hyperaldosteronism, extracellular volume depletion, and increased ADH levels in Klotho deficiency (107). However, Zhou et al. report higher NCC levels in Klotho+/- mice that also have hyperaldosteronism (105) and Andrukhova et al. report lower NCC levels in Klotho-/- mice (32). A few pieces of the puzzle with regard to Klotho, sodium handling, and blood pressure are still to be clarified. Another discrepancy with that mechanism would be that at 8-9 weeks of age, kl/kl mice were still found to have a lower systolic blood pressure than WT mice, without responding to spironolactone treatment (53).

Interventions in the development of hypertension

Zhou et al. detail that the increased salt sensitivity in kl/+ mice, manifested as an additional increase in blood pressure after 4 weeks of a 2% saline diet, could be reversed by selective CC chemokine receptor 2 (CCR2) inhibitors INCB3284 and RS102895 (105). However, baseline hypertension due to partial Klotho deficiency was unaltered. This indicates that the monocyte chemoattractant protein 1 (MCP-1)/CCR2 pathway may be involved in Klotho deficiency-induced increased salt sensitivity, but not in the initial development of hypertension. Targeting the increased aldosterone signaling present in Klotho deficiency using eplerenone, however, does seem to affect the Klotho decrease-induced hypertension, reducing blood pressure to WT levels (106). Also, both baseline hypertension and high-fat diet-induced exacerbation of hypertension in kl/+ mice could be normalized by AMPK activator AICAR (91).

Klotho overexpression and hypertension

Saito et al. first found that systolic blood pressure was reduced in OLETF rats after Klotho gene transfer (102). Klotho gene delivery has also been shown by Wang et al. to prevent the development of hypertension in SHR during the study period of 12 weeks after gene delivery (108). Klotho overexpression in SHR was associated with less oxidative stress and down-regulation of Nox2 protein expression and NADPH oxidase activity, indicative of an anti-oxidative state. In another study, Klotho overexpression normalized hypertension induced by 5/6th nephrectomy as well (109). A study using recombinant Klotho protein, however, did not find an effect on blood pressure in ApoE-/- mice after 3 weeks of treatment, nor did a study using SHR, 7 days after gene transfer (97, 103).

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Klotho and blood pressure regulation

Given the complex regulation of blood pressure, the findings of hypertension in (partial) Klotho deficiency and Klotho-induced reduction of blood pressure in spontaneous hypertension warrant a more elaborate background for discussion. Renal Klotho gene expression has been uniformly shown to be decreased in many experimental models of circulatory stress, including SHR (99, 108, 110-112), DOCA salt-sensitive hypertensive rats (99, 110), hypertensive/diabetic OLETF rats (99, 110, 113), angiotensin II infusion in rats (114), and ApoE-/- mice (115). The mechanism, however, behind this down-regulation is not clear and probably involves many different factors acting in concert, including angiotensin II, aldosterone, oxidative stress, and pro-inflammatory factors. It is unlikely that Klotho is down-regulated in a direct response to hypertension, since norepinephrine infusion in rats for 7 days did not down-regulate Klotho (116). Indirectly, however, long-term hypertension-induced renal damage probably does down-regulate Klotho expression.

Klotho has been shown to modulate many determinants of blood pressure, including the left ventricular cardiac muscle, which is prone to hypertrophy in Klotho deficiency and is protected by Klotho overexpression (16, 17, 117-119). Furthermore, the aortic valve exhibits hinge region calcification and fibrosis in Klotho deficiency (58, 87, 120, 121), and of course arterial resistance is increased in the setting of extensive arterial calcification. The endothelin system has been shown to be downstream of Klotho, since silencing of CNS Klotho using shRNAs in the cerebrospinal fluid potentiated cold-induced hypertension, which was prevented by concurrently silenced endothelin-1 (122). Endothelin plasma levels were later found to be down-regulated by Klotho overexpression, while concurrently preserving ETB receptor levels (123). The baroreflex blood pressure regulation mechanism in SHR has been shown to be impaired if Klotho is silenced and is restored by recombinant Klotho (124). In a similar study, recombinant Klotho or rosiglitazone administration, reverting down-regulation of Klotho, in streptozotocin-induced diabetes in rats was shown to improve the diabetes-impaired baroreflex (125).

Klotho and cardiovascular risk management

The observation that many of the medicines that are used to reduce the risk of cardiovascular events seem to influence Klotho expression is especially interesting. Many studies have identified a link between the renin-angiotensin-aldosterone system and Klotho. Klotho was shown to regulate CYP11B2 transcription factors SF-1 and ATF2, likely resulting in inhibition of adrenal aldosterone synthesis (106). Klotho also down-regulates the expression of angiotensinogen, renin, angiotensin converting enzyme, and angiotensin II receptor type 1, by blocking Wnt signaling and the consequent Wnt-induced up-regulation of the aforementioned RAAS genes (109). Conversely, angiotensin II has been shown to down-regulate Klotho even in the absence of hypertension (114). Direct or indirect angiotensin II-

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mediated down-regulation of Klotho has been demonstrated to be restored by ACE inhibitors (111, 112, 126-128), angiotensin II receptor antagonists (111, 112, 114, 126, 127, 129, 130), renin inhibitors (131), and aldosterone receptors antagonists (132). The pressor-independent nature of this effect is further supported by similar findings in renal tubular epithelial cells in vitro, while identifying RhoA activation as a possible pathway by which angiotensin II suppresses Klotho (133, 134). Similarly, statins have been shown to up-regulate Klotho, possibly through RhoA inactivation (134-136), whereas PPAR-γ agonists even cause a direct PPAR-γ-mediated increase in transcriptional activity of the Klotho gene (113, 125, 137-139).

Impaired angiogenesis and vasculogenesis

Impaired angiogenesis and vasculogenesis in Klotho deficiency

As noted before, kl/kl mice exhibit decreased VEGF expression and NO production in aorta (101), raising the question whether angiogenesis and vasculogenesis may be impaired in kl/kl mice. Indeed, Fukino et al. first described that heterozygous kl/+ mice display impaired angiogenesis in response to unilateral hind limb ischemia (140). Laser Doppler perfusion-assessed blood flow was significantly higher in WT mice between 7 and 14 days after induction of ischemia, after which the blood flow became statistically comparable again between groups (although the kl/+ mice never reached WT levels). Kl/+ mice also exhibited decreased capillary density 5 weeks after surgery. A similar experiment was described by Shimada et al., also performing unilateral hind limb ischemia, in WT, kl/+, and kl/kl mice (100). The authors describe significantly reduced blood flow in kl/+ mice as compared to WT mice, as well as in kl/kl mice as compared to kl/+ mice, between 3 days after surgery and the end of the study period (28 days after surgery). Capillary density was also significantly reduced in both kl/+ and kl/kl mice. Ex vivo aortic ring microvessel sprouting was also impaired in kl/kl mice and these mice had fewer EPC-like progenitor cells in both bone marrow and peripheral blood. Concurrent homologous bone marrow transplantation (WT to WT and kl/kl to kl/kl, which would be interesting heterologously) and hind limb ischemia revealed less engraftment of CD31+ or vWF+ donor cells after 14 days, suggestive of impaired vasculogenesis in Klotho deficiency. Furthermore, spontaneous limb amputation occurred more frequently in kl/kl animals than in WT animals. It should be noted that Klotho does not appear to have uniformly pro-angiogenic properties. For example, the retinal choroid layer of kl/kl mice is severely deformed with dilated blood vessels and Klotho may inhibit VEGF secretion from the retinal pigment epithelium, essentially exerting anti-angiogenic effects (141). In short, Klotho deficiency is associated with impaired angiogenesis and vasculogenesis, but the effects may differ per tissue or organ.

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Interventions in angiogenesis

There are two reports on interventions aimed at modulating angiogenesis in Klotho deficiency. Shimada et al. have performed unilateral hind limb ischemia in kl/kl mice with and without cerivastatin (s.c., 5 mg/kg/day for 28 days after surgery), thought to induce vasodilation and enhance NO synthase activity (100). Laser Doppler blood flow was increased as compared to untreated animals, as were capillary density, ex vivo aortic sprouting, and the number of EPCs in bone marrow and peripheral blood. Amputation rate was also significantly improved by cerivastatin treatment. Arima et al. found that VEGF stimulation can ameliorate the impaired angiogenesis of Klotho deficiency, which is further improved by antibody-mediated inhibition of pigment epithelium-derived factor (PEDF) (142). PEDF mRNA was found to be up-regulated in response to VEGF and PEDF administration was found to completely abolish VEGF-induced angiogenesis. It is unknown whether PEDF is up-regulated in kl/kl mice as compared to WT mice. Experiments assessing the effect of Klotho overexpression on angiogenesis have not been published to date.

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Table 2. Effects of interventions on other vascular phenotypes in models of Klotho deficiency

Intervention Animal model Effect on phenotype Reference Parabiosis Kl/+ and WT mice Improvement of endothelial

function Saito, 1998

Inhibition of aldosterone signaling

Kl/+ mice + eplerenone Prevention of arterial stiffening Chen, 2015

High-fat diet Kl/+ mice + high-fat diet Acceleration of development of arterial stiffening

Lin, 2016

AMPK activation Kl/+ mice + AICAR Prevention of arterial stiffening Lin, 2016 AMPK activation Kl/+ mice + high-fat diet +

AICAR Prevention of arterial stiffening Lin, 2016

High-fat diet Kl/+ mice + high-fat diet Acceleration of development of hypertension

Lin, 2016

AMPK activation Kl/+ mice + AICAR Prevention of hypertension Lin, 2016 AMPK activation Kl/+ mice + high-fat diet +

AICAR Prevention of hypertension Lin, 2016

Inhibition of aldosterone signaling

Kl/+ mice + eplerenone Normalization of blood pressure

Zhou, 2015

Salt loading Kl/+ mice + 2% saline drinking water

Aggravation of hypertension Zhou, 2015

CCR2 inhibition Kl/+ mice + high salt diet + INCB3284

Normalization of increased salt sensitivity, unaltered hypertension

Zhou, 2015

CCR2 inhibition Kl/+ mice + high salt diet + RS102895

Normalization of increased salt sensitivity, unaltered hypertension

Zhou, 2015

Statin treatment Kl/kl mice + unilateral hind limb ischemia + cerivastatin

Improvement in angiogenesis Shimada, 2004

VEGF treatment Kl/kl mice + unilateral hind limb ischemia + VEGF

Improvement in angiogenesis Arima, 2010

VEGF treatment + PEDF inhibition

Kl/kl mice + unilateral hind limb ischemia + VEGF + PEDF antibodies

Increased improvement in angiogenesis

Arima, 2010

VEGF treatment + PEDF Kl/kl mice + unilateral hind limb ischemia + VEGF + PEDF

Impaired angiogenesis Arima, 2010

AMPK, 5’ adenosine monophosphate-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide ribinucleotide VEGF, vascular endothelial growth factor; PEDF, pigment epithelium-derived factor.

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Endogenous vascular Klotho expression

Having assessed all reported in vivo effects, Klotho appears to be a very promising factor to target. A logical first step would be to assess whether the vasculature is best targeted by increasing serum levels of Klotho, or by up-regulating endogenous Klotho expression. This endeavor, however, will immediately encounter disputed and conflicting evidence. Having established that the effects of Klotho on the vasculature appear to be mediated predominantly by soluble Klotho, the potential existence of endogenous vascular Klotho expression is still the most controversial topic in the current field of vascular Klotho biology.

Endogenous Klotho in aorta and other arteries

The first hint at possible vascular Klotho expression can be found in the original study by Kuro-o et al. (1). A Northern blot for Klotho produced a faint band in mouse aorta that contrasted markedly with high renal and cerebral Klotho expression, being the faintest band of all organs that exhibit Klotho expression and being slightly exceeded by putative expression in colon and thyroid gland. A follow-up study by the same group, however, using RT-PCR on mouse aorta instead of Northern blotting, failed to show expression of alternatively spliced Klotho mRNA (143). The expression of alternatively spliced Klotho is usually predominant over membrane-bound Klotho mRNA in mouse by a factor 10 and the expression pattern is otherwise consistent with the expression pattern of membrane-bound Klotho mRNA. The first protein data consisted of induction of expression in aorta on WB at the same weight as renal Klotho after, but not before, Klotho gene transfer (114). Immunoreactivity with proteins of higher and lower molecular weight, however, was detected in aorta, heart, and liver, which may reflect the existence of different Klotho proteins, but may also reflect aspecific immunoreactivity of anti-rat IgG secondary antibodies on rat tissue lysates. Mice with Klotho promoter-driven X galactosidase expression revealed endogenous Klotho expression in renal distal tubules, parathyroid gland, and brain, as expected (95). Sino-atrial node Klotho expression was an interesting new finding in this study, but arterial Klotho expression was not observed. It is possible however, that the expression level is lower than could be detected by this method. This interpretation is endorsed by three studies from another group, who have only used RT-PCR on human aorta, generally detecting a low expression level of Klotho (144-146). This is in line with our own data, yielding just barely detectable levels of Klotho mRNA in various human arteries (147). However, in various other studies, Klotho protein and mRNA could not be detected in rat or mouse aorta using various techniques (71, 93, 108, 148, 149). Interestingly, RNA-Seq analysis of normal and uremic rat aorta did not yield values for Klotho above the commonly used threshold of 1 RPKM (1 read/kilobase transcript/million mapped

47

reads, roughly amounting to 1 transcript per cell) and reads were mostly mapped to introns, rather than exons (150). Another group reports RPKM values for Klotho of 0.1025 in normal rats, and 0.0042 in CKD rats, which would roughly correspond to 1 transcript per 10 and 250 cells, respectively (94). Our own group has also assessed vascular Klotho protein expression in human arteries, with negative results, concluding that the renal membrane-bound Klotho protein is not expressed in the vasculature (147). Furthermore, an extensive study by Lindberg et al. shows that Klotho protein is not detected in WT mouse arteries, nor in SM22α-Cre/KL-LoxP mice, which do not express the Klotho gene in smooth muscle cells (151). Klotho mRNA was detected in WT mouse aorta at a level several thousand-fold lower than in kidney and at a level that is similar in SM22α-Cre/KL-LoxP mice and β-actin-Cre/KL-LoxP mouse aorta. This suggests that vascular smooth muscle Klotho gene expression was likely already negligible to begin with in WT mice, since knocking it out systemically or selectively did not affect the expression levels. The lack of vascular abnormalities in these mice is also a strong argument against the existence of smooth muscle cell Klotho, at least if it were presumed to be involved in the prevention of medial calcification and intima hyperplasia. However, the absence of other precipitating factors, such as hyperphosphatemia and hypercalcemia, does not definitively preclude the possible emergence of phenotypic abnormalities if these conditions were to be met. The lack of baseline Klotho expression, however, renders this option unlikely. Another argument against vascular expression of membrane-bound Klotho is the lack of Egr-1 up-regulation upon acute stimulation with FGF23 in vivo, a response that is intact in the kidney and that is indicative of FGF23 signaling for which Klotho is known to be an obligate co-receptor (151). This experiment was repeated by our own group using a protocol of chronic FGF23 infusion, which also did not elicit Egr-1 up-regulation in aorta, but did so in kidney (147). Further downstream, ERK1/2 phosphorylation was not found in VSMCs upon stimulation with FGF23 (152).

The study by Lindberg et al. was apparently not sufficient to resolve the controversy, because the view that Klotho is expressed endogenously in arteries has remained widely prevalent. A number of authors show data indicative of vascular Klotho expression (detailed in Table 3) (94, 152-159). The expression patterns in these studies are very different and it is unclear whether Klotho expression is present at baseline and down-regulated in CKD or not down-regulated in CKD, or virtually absent at baseline and induced in calcified lesions. It is very likely that technical differences, including the usage of different antibodies, are chiefly responsible for these highly discrepant findings. Furthermore, only a few authors have investigated Klotho mRNA levels in arteries (152-155), some of which show Klotho mRNA levels that were 200- up to several thousand-fold lower than β2-microglobulin expression, which would be compatible with low Klotho mRNA expression levels discussed previously. Finally, the only two studies that do provide compelling evidence that at least some kind of Klotho-related protein is present in arteries were only very recently published. The first study, by Zhao et al., indicates that an unspecified Abcam antibody detects a band of unknown height on WB in WT mouse aorta, that is decreased in intensity in CKD, but partially restored by rapamycin and

48

that is not detected in Klotho-/- mouse aorta (57). The second study, by Lim et al., shows positively stained aorta and arteries and bands using WB of 116 kDa on the same tissues, with recombinant human Klotho at 116 kDa as well (160). Interestingly, the authors also used mass spectrometry and detected a number of Klotho-specific peptides in aorta and arteries. However, it cannot be excluded that there was contamination with soluble Klotho, which also contains the KL2 region from which the detected peptides derive.

Endogenous Klotho in smooth muscle cells

Many in vitro studies have addressed the question of endogenous smooth muscle cell Klotho expression, the first of which were Wang et al., who showed that neither Klotho mRNA, nor protein could be detected in rat aortic smooth muscle cells (RASMCs) before transfection with Klotho (108, 122, 161). Scialla et al. also did not find Klotho expression in human or mouse VSMCs (148) and our group detected no Klotho mRNA (above the detection threshold) in human SMCs (147). Other authors, however, do produce data indicative of substantial endogenous Klotho expression in SMCs (detailed in Table 4). A number of authors show that in human coronary SMCs, Klotho gene expression can be detected by RT-PCR (52, 53), whereas others detect a protein, usually of 116 kDa in size which was distinctly smaller than recombinant Klotho (94, 153, 156, 160). Functionally, silencing of Klotho was associated with an increased propensity towards calcification and increased Runx2 mRNA expression and Klotho expression could be restored by VDR agonists both in vitro and ex vivo (153). Another study found that anti-calcification effects of intermedin1-53 are mediated by Klotho (94). Intact signaling via the calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein 3 (RAMP3) complex and protein kinase A (PKA) was required for the inhibitory effect on vascular calcification, which resulted in up-regulation of Klotho. To what extent a vascular Klotho protein or kidney-derived Klotho mediates this effect, is not yet fully elucidated (162). Jimbo et al. detected a small amount of Klotho mRNA, but no protein in RASMCs (152). Interestingly, Zhao et al. detected a band at unspecified height on WB in HASMCs and BASMCs that is down-regulated by phosphate, up-regulated or restored by rapamycin or Klotho vector transfection, and targeting of which by siRNAs down-regulated the protein and increased calcification (57). These authors also detect Klotho mRNA that is down-regulated by phosphate and leucine-induced mTOR activation, but up-regulated by rapamycin.

49

Tabl

e 3

– Fi

ndin

gs w

ith re

gard

to K

loth

o ex

pres

sion

in a

rter

ial t

issue

Publ

icatio

n Kl

otho

exp

ress

ion

Spec

ies

Tiss

ue/ c

ell t

ype

Met

hods

An

tibod

y Re

mar

ks o

n ex

pres

sion

Posit

ive

cont

rol

Kuro

-o, 1

997

Low

(mRN

A)

Mou

se

Aort

a NB

Ye

s Sh

iraki

-Iida

, 19

98

No

Mou

se

Aort

a RT

-PCR

Ye

s

Mita

ni, 2

002

No

Rat

Aort

a W

B KM

2076

Yes

Take

shita

, 20

04

No

Mou

se

Full

mou

se

X-ga

l st

aini

ng i

n kl

-geo

m

ice

Yes

Dona

te-

Corr

ea, 2

011

Yes (

mRN

A)

Hum

an

Aort

a RT

-PCR

, qRT

-PCR

No

Nava

rro-

Gonz

áles

, 20

13

Yes (

mRN

A)

Hum

an

Aort

a RT

-PCR

, qRT

-PCR

No

Men

cke,

201

5 No

(p

rote

in),

Low

(m

RNA)

Hu

man

, m

ouse

Ar

terie

s (h

uman

, m

ouse

) W

B, IH

C, q

RT-P

CR

KM20

76,

SC-

2222

0, S

C-22

218,

AF

1819

Ye

s

Wan

g, 2

009

No

Rat

Aort

a RT

-PCR

, WB

AF18

19

Ye

s Fo

n Ta

cer,

2010

No

M

ouse

Ao

rta

qRT-

PCR

Yes

Lau,

201

2 No

M

ouse

Ao

rta

IHC,

qRT

-PCR

, WB

KM20

76

Ye

s Sc

ialla

, 201

3 No

M

ouse

Ao

rta

RT-P

CR

Yes

Lindb

erg,

20

13

No

(pro

tein

), Lo

w

(mRN

A)

Mou

se

Aort

a IH

C, W

B, q

RT-P

CR

KM20

76, K

M21

19

Ye

s

Lim, 2

012

Yes (

prot

ein)

Hu

man

M

esen

teric

art

ery

IHC,

WB,

qRT

-PCR

Ab

7502

3 De

crea

sed

in C

KD,

up-

redu

late

d by

VD

R ag

onist

s

No

Fang

, 201

3 Ye

s M

ouse

Ao

rta

qRT-

PCR,

IHC

AF18

19

Decr

ease

d in

CKD

Ye

s Fa

ng, 2

014

Yes

Mou

se

Aort

a qR

T-PC

R, IH

C AF

1819

De

crea

sed

in C

KD

Yes

Zhu,

201

3 Ye

s M

ouse

Ao

rta

qRT-

PCR,

WB,

IHC

Abca

m ?

No

ne

at

base

line,

up

-re

gula

tion

in

calci

fied

lesio

ns

Yes

50

Jimbo

, 201

3 Ye

s Ra

t Ao

rta

qRT-

PCR,

WB,

IHC

KM20

76

No ch

ange

in C

KD

No

Zhan

g, 2

014

Yes

Rat

Aort

a W

B ?

Decr

ease

d in

va

scul

ar

calci

ficat

ion

No

Ritt

er, 2

015

Yes

Rat

Aort

a IH

C AB

IN50

2138

De

crea

sed

in m

edia

in

CKD,

in

crea

sed

in

adve

ntiti

a in

CKD

Yes

Van

Venr

ooij,

20

14

Yes

Hum

an

Coro

nary

art

ery

qRT-

PCR,

IHC,

IF

KM20

76,

? Im

mut

opics

No

ne

in

norm

al

cells

, on

ly in

calci

fied

lesio

ns

No

Zhao

, 201

5 Ye

s M

ouse

Ao

rta

(mou

se)

WB

Abca

m ?

De

crea

sed

in

CKD,

in

crea

sed

by ra

pam

ycin

Ye

s

Lim, 2

015

Yes

Hum

an

Arte

ries

WB,

IHC,

MS

Ab69

208o

r Ab

1813

73

Ye

s

Dona

te-

Corr

ea, 2

015

Yes (

mRN

A)

Hum

an

Aort

a qR

T-PC

R

No

Ruko

v, 2

016

Not

abov

e cu

t-off,

no

t exo

nic m

RNA

Rat

Aort

a RN

A-Se

q, q

RT-P

CR

No

Chan

g, 2

016

Yes

(pro

tein

), no

(m

RNA)

Ra

t Ao

rta

RNA-

Seq,

WB,

IHC

Abca

m ?

De

crea

sed

in

CKD,

in

crea

sed

by

inte

rmed

in1-

53

Yes

Ide,

201

6 No

M

ouse

In

trar

enal

art

erie

s BR

ISH

Yes

NB, N

orth

ern

blot

; RT-

PCR,

rev

erse

tran

scrip

tase

pol

ymer

ase

chai

n re

actio

n; W

B, W

este

rn b

lot;

qRT-

PCR,

qua

ntita

tive

real

-tim

e po

lym

eras

e ch

ain

reac

tion;

IHC,

imm

unoh

istoc

hem

istry

; IF,

imm

unof

luor

esce

nce;

MS,

mas

s sp

ectr

omet

ry; B

RISH

, brig

ht-fi

eld

in s

itu h

ybrid

izatio

n; C

KD,

chro

nic k

idne

y di

seas

e; V

DR, v

itam

in D

rece

ptor

.

51

On the whole, it is likely that Klotho mRNA is expressed at a very low level in vascular cells and tissues (the low level rendering it occasionally undetectable). The expression level, however, is difficult to deduce from almost all reported data, given the prevalent tendency to express it in arbitrary units or strictly in a manner relative to baseline or control group levels. The conflicting data are mainly found at the protein level. Considering the general lack of positive/negative controls and validation experiments for most antibodies, there is currently not enough evidence to support the view that membrane-bound Klotho protein, as expressed in the kidney, is expressed at an appreciable level in the vasculature. It is striking that, whenever the protein size is reported or can be deduced, Western blot results often identify a protein of 116 kDa in size in vascular samples, but never the 130 kDa protein that would be expected, since both recombinant Klotho and endogenous renal Klotho are 130 kDa in size. It is certainly suggestive that many different antibodies raised against (a portion of) the Klotho protein exhibit immunoreactivity in the vasculature (disregarding the discrepancies in staining pattern). However, the protein(s) with which these antibodies react have not been characterized and the reported differences in size on Western blot analysis render it unlikely that it concern membrane-bound Klotho as expressed in the kidney. This is further attested by the finding that methods that do detect renal Klotho generally do not detect Klotho in the vasculature (147). The vascular epitopes may still concern proteins that are products of Klotho mRNA translation or that are otherwise related to Klotho (as may be the case with the proteins reported by Zhao et al. (57) and Lim et al. (160)). There is, however, no evidence to support the view that membrane-bound Klotho, as it expressed in the kidney, is expressed in smooth muscle cells, or that it has any functionality as a co-receptor for FGF23. There is, however, a lot of well-founded research that challenges the endogenous expression of membrane-bound Klotho in SMCs. There are many studies with conflicting data (even the studies that argue in favour of vascular Klotho expression are diametrically opposed in many ways). Therefore, well-validated studies with proper controls are required to assess whether Klotho is expressed in any shape or form in the vasculature and which epitopes are reactive with which anti-Klotho antibodies.

52

Tabl

e 4.

Fin

ding

s with

rega

rd to

Klo

tho

expr

essio

n in

smoo

th m

uscle

cells

.

Publ

icatio

n Kl

otho

ex

pres

sion

Cell

type

M

etho

ds

Antib

ody

Rem

arks

on

expr

essio

n Po

sitiv

e co

ntro

l W

ang,

201

0 No

RA

SMCs

W

B, R

T-PC

R AF

1819

Yes

Wan

g, 2

012

No

RASM

Cs

qRT-

PCR,

WB

AF18

19

No

Sc

ialla

, 201

3 No

HA

SMCs

, M

ASM

Cs

RT-P

CR

Yes

Men

cke,

201

5 No

(p

rote

in),

Low

(mRN

A)

HASM

Cs

WB,

IF, q

RT-P

CR

KM20

76,

SC-2

2220

Yes

Naka

no-K

urim

oto,

20

09

Yes

HCAS

MCs

RT

-PCR

No

Lim, 2

012

Yes

HASM

Cs

IF, W

B, q

RT-P

CR

Ab75

023

Decr

ease

d in

ure

mia

or

by T

NF-α

, up-

regu

late

d by

VD

R ag

onist

s No

Voel

kl, 2

012

Yes

HASM

Cs

RT-P

CR

No

Zhu,

201

3 Ye

s M

ASM

Cs

WB

Abca

m ?

In

crea

sed

by β

-gly

cero

phos

phat

e an

d as

corb

ic ac

id

Yes

Jimbo

, 201

3 Ye

s (m

RNA)

, No

(pro

tein

) RA

SMCs

qR

T-PC

R, W

B KM

2076

No

Zhao

, 201

5 Ye

s HA

SMCs

, BA

SMCs

W

B Ab

cam

?

Decr

ease

d in

CKD

, inc

reas

ed b

y ra

pam

ycin

Ye

s

Lim, 2

015

Yes

HASM

Cs

WB,

MS

Ab69

208o

r Ab

1813

73

Ye

s

Lin, 2

016

No

MAS

MCS

W

B, R

T-PC

R AF

1819

Yes

Chan

g, 2

016

Yes

HASM

Cs

WB,

qRT

-PCR

Ab

cam

?

Decr

ease

d by

indu

ctio

n of

cal

cifica

tion,

incr

ease

d by

in

term

edin

1-53

, in

term

edin

1-53

-med

iate

d an

ti-ca

lcific

ef

fect

s are

Klo

tho-

depe

nden

t

No

RASM

Cs, r

at a

ortic

smoo

th m

uscle

cel

ls; H

ASM

Cs, h

uman

aor

tic sm

ooth

mus

cle c

ells;

MAS

CMs,

mur

ine

aort

ic sm

ooth

mus

cle c

ells;

HCA

SMCs

, hu

man

cor

onar

y ar

tery

sm

ooth

mus

cle c

ells;

BAS

MCs

, bov

ine

aort

ic sm

ooth

mus

cle c

ells;

WB,

Wes

tern

blo

t; RT

-PCR

, rev

erse

tra

nscr

ipta

se

poly

mer

ase

chai

n re

actio

n; q

RT-P

CR, q

uant

itativ

e re

al-ti

me

poly

mer

ase

chai

n re

actio

n; IF

, im

mun

oflu

ores

cenc

e; M

S, m

ass s

pect

rom

etry

; TNF

-α,

tum

or n

ecro

sis fa

ctor

α; V

DR, v

itam

in D

rece

ptor

; CKD

, chr

onic

kidn

ey d

iseas

e.

53

Endogenous Klotho in endothelial cells

Although putative endothelial Klotho expression is not considered a topic as controversial as SMC Klotho expression, the data are almost equally discrepant. The lack of attention may be explained by the lack of positive staining results, since only two studies claim to have detected Klotho in the endothelium: in fetal vessels in human placenta (163) and in many arteries, including aorta and vasa vasorum, by Lim et al.(160). While IHC for Klotho on ECs has not been the primary focus of a study so far, we are not aware of any other author noting positive capillary staining in any tissue.

Endothelial cells, however, have been subjected to investigations in vitro. Xiao et al. have shown that by using a newly generated antibody specific for the 60 kDa cleaved KL1 internal repeat of Klotho and using purified recombinant KL1 as a control, it was possible to detect soluble Klotho in serum, but not in HUVEC lysate (164). Hamdi et al. detected low Klotho mRNA expression by RT-PCR in human endothelial cells that was increased after 2 weeks of stimulation with 1 mM captopril, but expression was only found in endothelial cells that expressed lower angiotensin II levels, likely de-repressing Klotho expression (165). Other authors describe similarly low levels of Klotho mRNA in ECs (33, 52). Kusaba et al. did not detect Klotho protein in AdLacZ-transfected HUVECs or conditioned medium, and only in Ad-Klotho-transfected HUVECs and conditioned medium. However, these authors observed a very weak interaction between TRPC1 and VEGFR2 in WT ECs (as opposed to in kl/kl ECs) thought to be stabilized by endogenously produced and subsequently shedded Klotho. These mechanistic studies, however, may indicate an extremely low (undetectable) endogenous endothelial Klotho protein production in ECs.

A number of authors, however, describe findings indicative of more substantial Klotho expression in ECs (detailed in Table 5). Klotho protein as well as mRNA were claimed to have been detected in HUVECs, often curiously as a 63-64 kDa protein, if specified (166-168). Somehow, Markiewicz et al. detect Klotho mRNA levels that are threefold higher than β2-microglobulin levels, a highly expressed gene(169), which stands in marked contrast to the expression levels detected by others that are around the detection limit of common PCR analysis. The most compelling argument for endothelial Klotho expression stems from the RNA interference experiment by Sun et al., which resulted in Klotho mRNA and protein (of unspecified size) down-regulation (170). Finally, endothelial progenitor cells (EPCs) were shown to express Klotho mRNA and protein (60 kDa), silencing of which potentiated (angiotensin II-induced) senescence and attenuated the beneficial effects of calcitonin gene-related peptide (171).

Taken together, most data indicate that endothelial cells, akin to smooth muscle cells, may express Klotho mRNA at an extremely low baseline level. Discrepancies arise, again,

54

Tabl

e 5.

Fin

ding

s with

rega

rd to

Klo

tho

expr

essio

n in

end

othe

lial c

ells.

Publ

icatio

n Kl

otho

ex

pres

sion

Tiss

ue/ c

ell t

ype

Met

hods

An

tibod

y Re

mar

ks o

n ex

pres

sion

Posit

ive

cont

rol

Ohat

a, 2

011

Yes

Plac

enta

l en

doth

eliu

m

IHC

SC-2

2218

Yes

Xiao

, 200

4 No

HU

VECs

W

B Se

lf-m

ade

Ye

s Ha

mdi

, 200

4 Ye

s EC

s RT

-PCR

Incr

ease

d in

cas

e of

low

er a

ngio

tens

in

II le

vels

No

Kusa

ba, 2

010

Yes

(mRN

A), N

o (p

rote

in)

HUVE

Cs

RT-P

CR, W

B

No

Naka

no-K

urim

oto,

20

09

Yes

HCAE

Cs

RT-P

CR

No

Zhou

, 201

0 Ye

s EP

Cs

RT-P

CR,

IF,

WB

Novu

s Bio

logi

cals

? In

crea

sed

by C

GRP

No

Carr

aced

o, 2

012

Yes

HUVE

Cs

qRT-

PCR,

W

B Ab

7502

3 De

crea

sed

durin

g ce

llula

r agi

ng a

nd LP

S tr

eatm

ent

No

Buen

día,

201

4 Ye

s HU

VECs

qR

T-PC

R,

WB

Calb

ioch

em (4

2350

0)

No

Lim, 2

015

Yes

Intim

a of

art

erie

s IH

C Ab

6920

8or A

b181

373

Ye

s Su

n, 2

015

Yes

HUVE

Cs

WB,

qR

T-PC

R Ab

cam

?

Incr

ease

d by

RTE

F-1

No

Xi

a, 2

016

Yes

HUVE

Cs

WB,

RT-

PCR

? Up

-regu

late

d by

pra

vast

atin

and

dow

n-re

gula

ted

by T

NF-α

. Also

not

det

ecte

d be

fore

ove

rexp

ress

ion.

Dec

reas

ed IL

-6

prod

uctio

n, a

lso if

indu

ced

by T

NF-α

.

No

55

Mar

kiew

icz, 2

016

Yes

HDM

ECs

WB,

qR

T-PC

R Th

erm

o Fi

sher

?

Decr

ease

d ex

pres

sion

afte

r Kl

otho

siR

NA

tran

sfec

tion,

in

crea

sed

expr

essio

n af

ter

Klot

ho

stim

ulat

ion,

de

crea

sed

mig

ratio

n (s

crat

ch

assa

y)

afte

r Klo

tho

siRNA

tran

sfec

tion,

FGF

R1

coul

d no

t be

co-

imm

unop

recip

itate

d,

decr

ease

in

M

MP9

, CD

31,

VCAM

1 ex

pres

sion

afte

r Klo

tho

silen

cing

No

HUVE

Cs, h

uman

um

bilic

al v

ein

endo

thel

ial c

ells;

ECs

, end

othe

lial c

ells;

HCA

ECs,

hum

an c

oron

ary

arte

ry e

ndot

helia

l cel

ls; E

PCs,

end

othe

lial

prog

enito

r ce

lls; H

DMEC

s, hu

man

der

mal

micr

ovas

cula

r en

doth

elia

l cel

ls; IH

C, im

mun

ohist

oche

mist

ry; W

B, W

este

rn b

lot;

RT-P

CR, r

ever

se-

tran

scrip

tase

pol

ymer

ase

chai

n re

actio

n; q

RT-P

CR,

quan

titat

ive

real

-tim

e po

lym

eras

e ch

ain

reac

tion;

IF,

im

mun

oflu

ores

cenc

e; L

PS,

lipop

olys

acch

arid

e; R

TEF-

1, re

late

d tr

ansc

riptio

nal e

nhan

cer f

acto

r 1; C

GRP,

cal

titon

in g

ene-

rela

ted

pept

ide;

MM

P9, m

atrix

met

allo

prot

eina

se

9; V

CAM

1, v

ascu

lar c

ell a

dhes

ion

mol

ecul

e 1.

56

predominantly at the protein level, and are mostly based on Western blotting results that show immunoreactivity with (a) smaller protein(s) of 60-64 kDa, the nature of which has not been characterized (166, 167, 171). While an age-related decrease in protein level (167) or down-regulation in uremia (172) are certainly expected of Klotho, these observations are not proof in their own right of the identity of the investigated protein, nor is down-regulation of Klotho mRNA by siRNAs without demonstration of subsequent down-regulation of protein (172). It is possible that these antibodies recognized a novel, smaller, soluble Klotho protein, similar to the antibody used by Xiao et al. that did not detect such a protein in HUVECs (164). Without proper controls and validation, however, there is no ground for such an assumption and compelling evidence in support of substantial and relevant Klotho protein expression by endothelial cells is currently lacking.

Effects of exogenous Klotho in vitro and ex vivo

Given the reported anti-ageing effects that Klotho exerts, identification of the exact mechanisms and involved signaling pathways could greatly increase our understanding of ageing on a cellular level. The effects of Klotho on the vasculature are manifold and the mechanisms are only starting to be elucidated. Although the effect of Klotho on ex vivo or in vivo vessels has not been investigated thoroughly, many studies have been performed focusing on the effects of Klotho on smooth muscle cells (SMCs) and endothelial cells (ECs) in vitro.

Effects of exogenous Klotho on vessels

A very recent study by Chen et al. has examined the use of Klotho-releasing nanoparticles on de-cellularized vascular matrix in tissue-engineered blood vessels in an attempt to improve the hypoxic microenvironment that is plagued by high concentrations of phosphate (173). It was found that 9/10 tissue-engineered blood vessels exposed to Klotho from nanoparticles were still patent without intima hyperplasia or thrombus, 6 months after engraftment in rat carotid arteries, as compared to 1/10 patent vessels in the control group, most of which had formed thrombus. Furthermore, exposure to Klotho ensured the formation of a full endothelial lining and an almost normal blood flow of 5.8 ml/min. These effects were attributed to inhibition of apoptosis, increased engraftment of endothelial precursor cells (EPCs), and a reduction in phosphate-induced toxicity in monocytes/macrophages. These data illustrate the potent effects of Klotho on vascular health. With regard to effects on vascular

57

function, there is only one study in murine aortic rings that shows, counterintuitively, that Klotho induces vasoconstriction that could be abolished by ROS scavenger dimethylthiourea and ERK inhibitor U0126 (174). However, Klotho attenuated FGF23-induced vasoconstriction and up-regulated expression of p-eNOS and inducible (i)NOS, suggesting that Klotho exerts vasodilatory effects. This hypothesis was corroborated by the findings that phosphate-induced vasoconstriction and phenylephrine-induced vasoconstriction were converted to relaxation after addition of Klotho, which was abolished after treatment with L-NAME and by removal of the endothelium. Part of these data are challenged, however, by experiments by Lindberg et al., who could not detect an effect of FGF23 on vascular function (151). The vasodilatory effects of Klotho owing to increased NO production, though, are in line with other studies on vascular function (72, 102).

Effects of exogenous Klotho on smooth muscle cells

As in many other cell types, Klotho was found to protect SMCs against induced oxidative stress. Klotho gene transfer in rat SMCs dose-dependently reduces protein expression of Nox2, a subunit of the major membrane-bound ROS producing NADPH oxidase (161). This was likely a reflection of a post-translational mechanism, since Nox2 mRNA levels were unaltered. Klotho gene transfer reduced both basal and angiotensin II-induced levels of superoxide, 4-HNE (a by-product of lipid oxidation) and apoptosis. It was found that Klotho gene transfer up-regulated intracellular cAMP levels and protein kinase A (PKA) activity, inhibition of which also abrogated the Klotho-mediated down-regulation of Nox2 protein. Curiously, Six et al. report a slight increase in superoxide production in human SMCs upon stimulation with 1.6 nM of Klotho (174).

In another study, 0.4 nM of soluble Klotho was found to inhibit phosphate uptake and phosphate-induced mineralization in rat SMCs (3). This effect was associated with maintenance of differentiation of the contractile smooth muscle cell phenotype because Klotho reduced SMC Pit1 and Pit2 gene expression levels, reduced Runx2 gene and protein expression levels, and maintained SM22α gene and protein expression levels. Similarly, Zhao et al. found that Klotho gene transfer in bovine SMCs increased soluble Klotho levels in the supernatant, reduced the phosphate-induced increase in calcium content, as well as Cbfa1 and Msx2 mRNA levels, while increasing SMC differentiation genes α-SMA and SM22α (57). Another study found that Klotho dose-dependently prevents β-glycerophosphate-induced calcification of rat VSMCs after 12 days, reaching control calcium content levels by stimulation with 50 ng/mL Klotho or with higher concentrations (175). This effect was accompanied by normalization of disturbed Runx2, BMP2, αSMA, and β-catenin levels, but could be nullified by down-stream activation of Wnt signaling by addition of GSK3 inhibitor LiCl, indicating that one mechanism of Klotho-mediated inhibition of VSMC calcification involves inhibition of Wnt

58

signaling. Scialla et al., however, did not find an effect of Klotho on SMC calcium and phosphate uptake (148). Finally, Jimbo et al. used Klotho gene transfer in human SMCs and found that induction of membrane-bound Klotho expression could establish FGF23 signaling, which was FGFR1-dependent, facilitated phosphate-induced calcification, and induced osteoblastic transformation (152). In normal VSMCs, though, in which Klotho protein was not detected, FGF23 did not induce FGF23 signaling or calcification. The lack of observed FGF23 effects in this study in non-transfected cells may be due to de-differentiation in vitro, or the observed FGF23 effects in transfected cells may be non-physiological.

Using a different approach, Lin et al. immunoprecipitated Klotho from serum to study the effects of partial Klotho deficiency on murine smooth muscle cells (91). They found that partial Klotho depletion in combination with high cholesterol decreased pAMPKα levels, increased collagen-I, and decreased liver kinase B1 (LKB1) protein and phosphorylation levels, but not calcium/calmodulin-dependent protein kinase (CaMKK) α or β. All of these changes could be rescued by AICAR treatment. The effects of exogenous Klotho on smooth muscle cells are summarized in Figure 3.

Effects of exogenous Klotho on endothelial cells

On the whole, the effects of Klotho on ECs in vitro have been investigated more thoroughly than the effects on SMCs. As in SMCs, Klotho protein or gene transfer was shown to increase cAMP levels, which up-regulated PKA activity (and, interestingly, ACE activity) in HUVECs (176, 177), but, specifically in HUVECs, not PKC activity (176). As in SMCs, Klotho protein-mediated increase in cAMP levels in ECs led to activation of anti-oxidative systems, such as up-regulation of Mn-SOD, partially through increased NO production via the cGMP/PKG pathway. It was found that Klotho indeed decreased angiotensin II-induced ROS production in HUVECs as well (178). Along the same line, apoptosis induced by H2O2 in HUVECs was decreased by 20 nM Klotho protein (with concurrently decreased caspase 3 and caspase 9 activity). The extent of the anti-apoptotic effects of Klotho, however, is not limited to counteracting oxidative stress, since ROS-independent, etoposide-induced apoptosis was also inhibited by Klotho (179). Furthermore, induction of senescence was also prevented by Klotho protein in HUVECs, at least in part upstream of the p53/p21 pathway. Another study also found that related transcriptional enhancer factor (RTEF)-1 protects against oxidative stress-induced senescence in HUVECs by inducing Klotho expression on a transcriptional level, while silencing of Klotho abrogated the protective effect and caused subsequent down-stream down-regulation of p53 and p21 (170). These results were confirmed in a later study that also discovered that the Klotho-induced effects that protect against p53/p21-mediated senescence and caspase 3/caspase 9-mediated apoptosis were abrogated by ERK and MEK inhibitors (180). This is indicative of involvement of the mitogen- activated protein (MAP) kinase pathway and via

59

Figu

re 3

. The

effe

cts

of K

loth

o on

sm

ooth

mus

cle c

ells.

Klo

tho

redu

ces

the

gene

ratio

n of

ROS

by

the

NADP

H ox

idas

e, r

educ

es t

he t

rans

crip

tion

of

oste

ocho

ndro

geni

c ge

nes,

indu

ces t

he tr

ansc

riptio

n of

smoo

th m

uscle

cel

l-spe

cific

gene

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ther

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inhi

bits

ost

eoch

ondr

ogen

ic sig

nalin

g, m

aint

ains

the

cont

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ile sm

ooth

mus

cle ce

ll di

ffere

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ted

stat

e an

d co

ntra

ctili

ty, a

nd p

reve

nts v

ascu

lar c

alcif

icatio

n.

60

an unknown mechanism, Klotho was shown to induce ERK and MEK phosphorylation within minutes. Similarly, indoxyl sulfate (IS)-induced ROS production, decrease in NO and eNOS phosphorylation, and cellular injury could be inhibited in HUVECs by 0.2 or 0.4 nM Klotho protein (181). This effect was at least in part due to decreased p38 MAPK phosphorylation and subsequent NFκB translocation to the nucleus and could be abolished by respective p38 MAPK and NFκB inhibitors. In similar experiments, Buendía et al. claim that exogenous Klotho has similar beneficial effects on HUVECs in uremia. However, they did not use Klotho protein, but a 19 amino acid Klotho peptide that was once the immunogen for antibody Ab75023, rather than representing a site of established biological activity, and which constitutes roughly 1.8% of the full Klotho amino acid backbone (172). In another study, the same group claims that the same peptide attenuates TNFα-induced increase in, but not baseline levels of, ROS production and apoptosis in HUVECs (167). Chen et al. found that Klotho reversed in an ERK-dependent manner the decrease in proliferation, increase in apoptosis, and decrease in NO production, induced in HUVECs by serum of CKD patients with secondary hyperparathyroidism (182). Markiewicz et al. found that Klotho may also stimulate EC migration, although the concentration was not disclosed (169). Finally, Six et al. did not find an effect of Klotho protein on baseline ROS levels in HUVECs (174).

Aside from promoting cellular viability, Klotho has also been shown to suppress the expression of adhesion molecules and other pro-inflammatory factors in ECs. TNF-α-induced up-regulation of adhesion molecules ICAM-1 and VCAM-1 in vitro (HUVECs) and ex vivo (rat aorta) can be partially inhibited by 0.2 nM Klotho protein via inhibition of IκB phosphorylation and NFκB gene transcription (183). Functionally, this resulted in reduced adhesion of monocytes to HUVECs in vitro. In addition, Yang et al. report that in HUVECs, Klotho also reduces release of monocyte chemoattractant protein (MCP)-1, a pro-inflammatory factor that is also NFκB-mediated (181). In an elaborate study, Liu et al. found that intracellular Klotho protein, after Klotho gene transfer in HUVECs, could bind directly to retinoid acid-inducible gene-1 (RIG-1), thereby preventing RIG-1 multimerization (166). The result was inhibition of RIG-1-induced expression of IL-6 and IL-8 via suppression of NFκB, which also translated to attenuation of cellular senescence. Although expression of the full Klotho protein certainly blocked RIG-1-induced effects, specifically the intracellular KL1 sequence was obligatory, lacking the transmembrane domain, the signal peptide, and the KL2 repeat. Although Klotho was recently demonstrated to exert more intracellular functions (49, 184), in what form Klotho is physiologically present intracellularly, in which cell types, and whether this includes endothelial cells, remains to be determined.

Although extensive research has already yielded a lot of information on downstream signaling pathways, a specific receptor for Klotho has never been identified. It was demonstrated recently, however, by Takenaka et al. that a Klotho-IgG hybrid detected by anti-IgG-FITC could be visualized bound to endothelial cells in kidney sections, indicating the presence of an endothelial receptor for Klotho (185). Although not sufficiently explanatory for the effects on the abovementioned signaling cascades, direct interaction between Klotho and TRPC1 and

61

VEGFR2 has been observed, constituting another pathway by which Klotho protects the endothelium, by inhibiting VEGF-mediated calcium influx (33). Specifically, the KL2 internal repeat of Klotho was found to bind to the fifth loop of TRPC1 and the sixth and seventh Ig domains of VEGFR2, as assessed by mutational analysis and immunoprecipitation, and fluorescence resonance energy transfer microscopy. This interaction leads to internalization of the TRPC1/VEGFR2 complex, thereby preventing VEGF-mediated calcium influx, which in turn prevents overactivation of Ca2+-dependent enzymes, such as µ-calpain and caspase 3. Kl/kl ECs stimulated with VEGF indeed display a sustained TRPC1-mediated Ca2+ influx, leading to increased µ-calpain activity, which leads to decreased aII-spectrin, p120-catenin, and VE-cadherin levels, and to increased caspase 3 activity and apoptosis. These effects could be abolished by TRPC1 silencing and µ-calpain blockade by acetyl-leucyl-leucyl-norleucinal (ALLN). These data also illustrate that Klotho deficiency (be it endogenous or systemic) affects endothelial cells, whether due to lack of expressed protein or as an effect of having been primed by constitutive systemic Klotho deprivation, remains to be determined. Klotho overexpression was employed in HUVECs in this study, which leaves it unclear whether membrane-bound Klotho or soluble Klotho binds to TRPC1/VEGFR2. Because overexpressed Klotho was secreted into the medium, exogenously added soluble Klotho readily produces the effects under investigation, and no native membrane-bound Klotho protein was detected, soluble Klotho was determined to be the likely mediator. The effects of Klotho on endothelial cells are summarized in Figure 4. An interesting point that has not been given a lot of attention, was raised by Hu et al. and concerns the mystery of the manner in which soluble, serum-derived Klotho is capable of exerting effects on target cells in vivo that are physically behind the endothelial barrier (186). Almost all cell types are known to have a very low basal expression level of Klotho mRNA, but only very few cell types express detectable amounts of Klotho protein and the effects on other cells are predominantly dependent on soluble Klotho. Similarly, it is still a mystery how Klotho is excreted into the urine at the level of the proximal tubule, which has long been speculated to involve transcellular transport of Klotho protein (39). This was recently substantiated by Hu et al. in a series of experiments in which labeled Klotho is shown to be transported through proximal tubules (187). The molecular basis for this mechanism is yet to be elucidated, but Klotho would be required to pass through the endothelium first. It has indeed been observed that Klotho somehow traverses the endothelial barrier in the lung, as labeled recombinant Klotho could be detected in extravascular cells 20-30 minutes after intravenous injection (18). The molecular mechanism responsible for the transcellular transportation of Klotho is currently an unresolved chapter in Klotho biology.

62

Figu

re 4.

The

effe

cts o

f Klo

tho

on e

ndot

helia

l cel

ls. K

loth

o re

duce

s the

VEG

F-in

duce

d ca

lcium

influ

x by p

rom

otin

g end

ocyt

osis

of th

e TR

PC1/

VEGF

R2 co

mpl

ex.

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inhi

bits

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ract

ivat

ion

of c

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m-d

epen

dent

enz

ymes

, su

ch a

s ca

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and

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lpai

n, p

rote

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g th

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ll fro

m a

popt

osis

and

prot

ectin

g th

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doth

elia

l int

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y by

pre

vent

ing

cleav

age

of p

120-

cate

nin

and

subs

eque

nt E

-cad

herin

deg

rada

tion.

Klo

tho

also

pre

vent

s apo

ptos

is an

d se

nesc

ence

via

in

hibi

ting

the

p53/

p21

casp

ase

9/ca

spas

e 3

path

way

and

redu

ces r

eact

ive

oxyg

en sp

ecie

s via

indu

ctio

n of

NO

synt

hesis

and

Mn-

SOD.

Klo

tho

prev

ents

NF-

κB n

ucle

ar tr

anslo

catio

n by

inhi

bitin

g Iκ

B ph

osph

oryl

atio

n an

d p3

8 M

APK

phos

phor

ylat

ion,

via

unk

now

n m

echa

nism

s. La

stly

, Klo

tho

bind

s RIG

-1 a

nd d

own-

regu

late

s man

y ge

nes t

hat p

rom

ote

infla

mm

atio

n an

d le

ukoc

yte

adhe

sion.

63

Conclusion and future perspectives

Klotho deficiency causes a number of vascular phenotypes, including vascular calcification, intima hyperplasia, endothelial dysfunction, arterial stiffening, hypertension, and impaired angiogenesis and vasculogenesis. With regard to the molecular, histological and functional aspects that have been investigated so far, Klotho deficiency appears to constitute a solid model for human vascular ageing.

The pathogenesis of these processes has not been fully elucidated, but disturbed mineral homeostasis, induction of osteochondrogenic signaling, and cellular senescence have been implicated, as well as reduced NO production, µ-calpain overactivation via sustained TRPC1 activity, and increased PAI-1 levels. In addition to phosphate, these latter factors that influence cellular senescence and apoptosis are likely also very important in mediating the development of vascular calcification. Although phosphate may also be causally involved in inducing senescence, direct inhibition of insulin/IGF-1 signaling by Klotho binding to IGF1R, as well as inhibition of µ-calpain overactivation by Klotho binding to TRPC1/VEGFR2, points to additional mechanisms being at play. Although vascular calcification is relatively well-studied in Klotho deficiency, the contribution of anti-calcification mechanisms is still largely unclear. It would be very interesting to delineate whether anti-calcification mechanisms, such as fetuin A, are up-regulated or down-regulated in calcified lesions and whether double knockouts exhibit less or more calcification than Klotho-/- mice. Intima hyperplasia in Klotho deficiency is especially poorly studied, as no detailed histological or molecular description is available in current literature, let alone studies in which treatment are tested. The link between Klotho and angiogenesis also merits further study. Similary, arterial stiffening in Klotho deficiency has only very recently gained attention. It is currently unknown whether Klotho overexpression may protect against the development of arterial stiffening, however, since Klotho protects against fibrosis in many other organs and tissues, this may very well prove to be the case. Overall, there has been a tendency to studying Klotho+/- mice in addition to Klotho-/- mice, which offer several advantages, including a less severe (but still readily inducible) phenotype of vascular calcification and tolerance to anaesthesia and surgery. Heterozygosity for Klotho may be a more appropriate model for human ageing, in which Klotho levels decline, but are generally not absent.

The current controversy surrounding vascular Klotho expression remains to be solved. Considering all available data, we conclude that membrane-bound Klotho, as expressed in the kidney, is not expressed in arteries. However, this is only part of the puzzle, as it does not explain all positive data on vascular Klotho expression.

Both in smooth muscle cells and in endothelial cells, a lot of pathways have been studied that may contribute to the effect of Klotho on these cell types. A number of unresolved issues still

64

stands out, however, including the identification of (a) Klotho receptor(s) and elucidation of the mechanism of transcytosis through the endothelium and other cell types.

Given the potency of Klotho in preventing and reversing severe vascular phenotypes, both in Klotho deficiency and in other disease models, it may be worthwhile to investigate Klotho further as a potential therapeutic agent in cardiovascular disease.

65

Acknowledgments

This work was supported by a consortium grant from the Dutch Kidney Foundation [NIGRAM, CP10.11] (PIs: P.M. ter Wee, M.G. Vervloet (VU Medical Center, Amsterdam), J.G. Hoenderop, R.J. Bindels (Radboud University Medical Center, Nijmegen), and G.J. Navis, M.H. de Borst, J.L. Hillebrands (University Medical Center Groningen, Groningen)) and the UMCG GSMS MD/PhD program.

66

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Part I

Vascular and renal Klotho

expression

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