University of Groningen
Klotho in vascular biology
Mencke, Rik
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Mencke, R. (2018). Klotho in vascular biology. Rijksuniversiteit Groningen.
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Chapter 10
Characterization of vascular function in
Klotho deficiency
R. Mencke A. van Buiten N. Stege O.W. Moe H. Buikema J.L. Hillebrands Manuscript in preparation310
Abstract
Endothelial dysfunction is an important risk factor for cardiovascular disease. It is generally found in conditions that also feature a deficiency in Klotho, a renal anti-ageing protein. Conversely, full Klotho knockout mice have long been known to develop severe vascular abnormalities and heterozygous mice have been found to have endothelial dysfunction. The goal of this study was to characterize vascular function in Klotho-/- mice.
We used aortic rings from Klotho-/-, Klotho+/-, and WT mice to determine the relaxation
response to acetylcholine after pre-contraction with phenylephrine and thromboxane A2 receptor agonist U46619. We determined phenylephrine and U46619 dose-response relationships. Finally, we incubated Klotho-/- and Klotho+/- rings with recombinant Klotho to
determine whether exogenous Klotho affected subsequent acetylcholine-induced vasodilation.
Klotho-/- mice displayed marked endothelial dysfunction compared to Klotho+/- and WT mice.
Phenylephrine-induced pre-contraction, however, was also significantly higher in Klotho
-/-rings. This was not the case with U46619 and Klotho-/- rings still displayed a markedly impaired
dilatory response. Neither in young (8-12-week-old), nor in old (52-55-week-old) Klotho
+/-mice could endothelial dysfunction be observed. Incubation with recombinant Klotho improved acetylcholine-induced relaxation of Klotho-/- and Klotho+/- rings, albeit not
significantly.
In conclusion, this is the first study to characterize vascular function in Klotho-/- aortic rings
and we confirm the presence of endothelial dysfunction in Klotho deficiency. There was also an increase in sensitivity to phenylephrine, as may occur in ageing. We could not confirm endothelial dysfunction in Klotho+/- mice and we provide indications of direct Klotho effects
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Introduction
Endothelial dysfunction is a common finding in ageing, hypertension, and chronic kidney disease (CKD) and it is thought to be involved in the early pathogenesis of atherosclerotic cardiovascular disease (1-5). These conditions are all known experimentally to be accompanied by various degrees of deficiency in Klotho (6-8), which is considered a renal anti-ageing protein (9, 10). Klotho is a type I transmembrane protein expressed primarily in distal convoluted epithelial cells (11, 12), from which it is also cleaved, giving rise to soluble Klotho in blood and urine (13), which then exerts its effects on target tissues.
Similar to CKD patients, which are progressively Klotho-deficient, with endothelial dysfunction and related comorbidities, it has been reported that partial deficiency of Klotho in mice results in endothelial dysfunction (14-16), which comports with the coincident decline in renal function and development of hypertension in these mice (17, 18). This renders Klotho a potentially causal factor in the development of both endothelial dysfunction and associated vascular abnormalities and end-organ damage. Profound effects on the vasculature are attributed to Klotho, since its absence leads to a phenotype of vascular calcification (9, 19, 20), arteriolar hyalinosis, and endothelial hyperpermeability (21) in full Klotho deficiency, and to a phenotype of hypertension (17, 18), and arterial stiffening (22-24) in partial Klotho deficiency. Endothelial dysfunction could play a role in the pathogenesis of many of these vascular conditions that also occur in humans during ageing, and especially in CKD. Promisingly, overexpression of Klotho has been shown to prevent endothelial dysfunction (15), but it is unknown whether this effect is directly mediated by soluble Klotho. As there have so far been no follow-up reports to initial indications of endothelial dysfunction in partial Klotho deficiency and vascular function in conduit arteries from Klotho-/- mice have so far not been
examined, the aim of this study was therefore to further characterize endothelial dysfunction in the complete absence of Klotho and to explore the direct effects of exogenous Klotho on endothelial dysfunction.
Materials and Methods
Animals
Klotho+/- mice were a generous gift from dr. Joost Hoenderop (Department of Physiology,
Radboud University Medical Center, Nijmegen, The Netherlands) and Klotho-/-, Klotho+/-, and
WT mice were bred at the University Medical Center Groningen. Animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
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Klotho-/- mice were 8-11 weeks old, Klotho+/- mice were 12 weeks old, and WT mice were
8-13 weeks old at the time of the baseline experiments. Vascular function in old Klotho+/- and
WT mice was also measured at 52-55 weeks of age and rings from 90-week-old Klotho+/- mice
were used for stimulation with recombinant Klotho.
Vascular function measurements
The thoracic aorta was removed after termination by cardiac puncture under isoflurane anaesthesia. Vascular ring preparations of approximately 2.5 mm were prepared and mounted on a small vessel wire myograph (Multi Myograph 610 M, Danish Myo Technology, Aarhus, Denmark) connected to a force displacement transducer, in an organ bath with Krebs solution bubbled with carbogen at 37 °C. A computer-assisted normalization protocol was used to pre-stretch rings to 90% of the diameter mimicking a transmural pressure of 100 mmHg, as described previously (25). Rings were allowed to equilibrate for 1 h after which they were primed and checked for viability by repeated stimulation with 60 mM KCl (three times with intermediate washings and re-stabilization). Subsequently, rings were pre-constricted with phenylephrine (PE, 1×10-6 M) or U46619 (3.0×10-8 M) and investigated for endothelial
dysfunction by cumulative stimulation with acetylcholine (ACh, 0.1 nM - 10 µM), followed by the administration of a single high dose of sodium nitroprusside (SNP, 0.1 mM) to induce maximal endothelium-independent relaxation. Phenylephrine-induced contraction curves (0.1 nM - 30 µM) and U46619-induced contraction curves (0.1 nM – 1 µM) were additionally generated in a separate or in the same set of rings after washouts, followed by post-constriction with 60 mM KCl to determine maximal receptor-independent contraction in the same setting. Recombinant mouse Klotho (kindly provided by dr. Orson Moe, UT Southwestern, Dallas, USA) was used at a concentration of 0.4 nM for pre-incubation for 2 hours at 37 °C, in addition to during acetylcholine stimulation. PE, U46619, SNP, and ACh were from Sigma-Aldrich (USA).
Statistical analysis
Ring preparations producing less than 0.5 mN force in response to constriction stimuli were excluded from analysis. ACh- and SNP-induced relaxation responses were expressed as a percentage of pre-constriction. PE- and U46619-induced contraction responses were expressed as a percentage of maximal contraction to 60 mM KCl given at the end of the protocol. Distribution of data was assessed using the Kolmogorov-Smirnov test. Normally distributed data were compared using ANOVA (followed by Bonferroni’s post-hoc correction) or Student’s t test and non-normally distributed data were compared using the Kruskal-Wallis (followed by Dunn’s post-hoc correction) or Mann-Whitney U test, depending on the number of groups. Paired data (rings from the same mouse incubated with PBS or with Klotho) were compared using a paired t test or Wilcoxon signed rank test, depending on the distribution.
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GraphPad Prism version 5.0 (GraphPad Software, USA) was used for statistical analysis and a
p value < 0.05 was considered significant.
Results
To assess whether Klotho deficiency induces endothelial dysfunction, we used aortic rings from Klotho-/-, Klotho+/-, and WT mice for ex vivo vascular function measurements. After
pre-constriction with phenylephrine (PE), incubation with increasing concentrations of acetylcholine (ACh) revealed that Klotho-/- rings displayed impaired relaxation compared to
Klotho+/- and WT rings (Figure 1A). Notably, there was no difference between Klotho+/- and
WT rings. However, even though force exerted by all rings was similar when repeatedly
Figure 1. Klotho-/- aortic rings display impaired endothelium-mediated acetylcholine-induced dilation after
pre-constriction with phenylephrine (PE). (A) KCl responses (mean ± SD) from rings depicted in (C). (B) Phenylephrine responses (mean ± SD) from rings depicted in (C). * p < 0.05, ** p < 0.01. (C) Responses from Klotho-/- (N = 6 rings
from 4 mice), Klotho+/- (N = 7 rings from 4 mice), and WT (N = 9 rings from 5 mice) aortic rings to acetylcholine
after pre-constriction with phenylephrine (mean ± SEM). * p < 0.05 (Klotho-/- vs. Klotho+/-). (D) Sodium
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incubated with KCl (Figure 1B), the subsequent PE-induced pre-contraction was significantly stronger in Klotho-/- rings (Figure 1C). Incubation with sodium nitroprusside (SNP) resulted in
complete relaxation of WT, Klotho+/-, and Klotho-/- rings (Figure 1D).
To assess whether the relative difference between rings in relaxation could be partially attributed to the difference in PE-induced pre-contraction levels, we performed another series of experiments employing thromboxane A2 receptor agonist U46619 to induce pre-constriction. Comparing Klotho+/- and Klotho-/- rings, we again found that Klotho-/- rings
displayed a marked dilatation impairment (Figure 2A). KCl incubations again produced similar contractions (Figure 2B) and, unlike with PE, there was no significant difference between Klotho-/- and Klotho+/- rings in U46619-induced pre-contraction (Figure 2C). SNP responses
after U41669 pre-contraction were similar across genotypes, albeit incomplete (Figure 2D). These latter data using U41669 as a means to induce comparable pre-constriction seems to preclude the possibility that the attenuated relaxation response to
Figure 2. Klotho-/- aortic rings display impaired endothelium-mediated acetylcholine-induced dilation after
pre-constriction with U46619. (A) KCl responses (mean ± SD) from rings depicted in (C). (B) U46619 responses (mean ± SD) from rings depicted in (C). (C) Responses from Klotho-/- (N = 6 rings from 3 mice) and Klotho+/- (N = 8 rings
from 4 mice) aortic rings to acetylcholine after pre-constriction with U46619 (mean ± SEM). * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Sodium nitroprusside (SNP) responses (mean ± SD) from rings depicted in (C).
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ACh in Klotho-/- rings might have been the result of functional antagonism rather than
indicative of impaired endothelial dysfunction. Collectively, the data indicate that Klotho
-/-mice, but not Klotho+/- mice, display endothelial dysfunction.
To confirm the above described differences in PE-induced pre-constriction levels, we additionally assessed per se contractility to PE and U46619 by generating full concentration-response curves to PE and U46619. While concentration-responses to KCl, albeit after a single high dose, were similar between the groups, again contraction responses to PE displayed a with a higher maximal contraction (Figure 3A), suggesting that PE in Klotho-/- aortic rings exhibits a higher
efficacy (rather than potency), as compared to Klotho+/- and WT rings (Figure 3A), although
this difference did not reach statistical significance.U46619 dose-response curves were similar across genotypes (Figure 3C) and KCl post-contractions were also similar after both PE and U46619 stimulations (Figure 3B, D).
Figure 3. Phenylephrine (PE) and U46619 dose-response curves. (A) Responses from Klotho-/- (N = 6 rings from
5 mice), Klotho+/- (N = 8 rings from 5 mice), and WT (N = 11 rings from 5 mice) aortic rings to phenylephrine
relative to the post-constriction KCl response (mean ± SEM). (B) KCl responses from rings depicted in (A). (C) Responses from Klotho-/- (N = 3 from 3 mice), Klotho+/- (N = 3 from 3 mice), and WT (N = 3 from 3 mice) aortic
rings to U46619 relative to the post-constriction KCl response (mean ± SEM). (D) KCl responses from rings depicted in (C).
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To assess whether Klotho+/- mice develop endothelial dysfunction at a later age, we repeated
the vascular function measurements in mice that were 1 year old. However, neither with PE as pre-constrictor (Figure 4A), nor with U46619 (Figure 4C) did we observe a difference between Klotho+/- and WT rings. PE- and U46619-induced pre-contractions were similar also
at this age (Figure 4B, D).
Finally, to investigate whether soluble Klotho is capable of directly and acutely rescuing endothelial dysfunction, we incubated Klotho-/- aortic rings with 0.4 nM recombinant mouse
Klotho (a kind gift from Dr. Orson Moe, UT Southwestern University, Dallas, USA) or PBS for 2 hours at 37 °C and during incubation with ACh. There was an increase in ACh-induced relaxation in rings incubated with Klotho (Figure 5A), although this difference was not significant. Note that this set of experiments was performed separately from the experiments in Figure 2A and that the acetylcholine response after U46619 pre-contraction
Figure 4. Klotho+/- aortic rings from 1-year-old mice do not display impaired endothelium-mediated
acetylcholine-induced dilation after pre-constriction with phenylephrine (PE) or U46619. (A) Responses from Klotho+/- (N = 12 rings from 6 mice), and WT (N = 11 rings from 6 mice) aortic rings to acetylcholine after
pre-constriction with phenylephrine (mean ± SEM). One spasmic ring was excluded. (B) Phenylephrine responses (mean ± SD) from rings depicted in (A). (C) Responses from Klotho+/- (N = 12 rings from 6 mice) and WT(N = 10
rings from 5 mice) aortic rings to acetylcholine after pre-constriction with U46619 (mean ± SEM). Two spasmic rings were excluded. (D) U46619 responses for rings depicted in (C).
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in this set of experiments was much weaker. Similarly, using rings from old (90-week-old) Klotho+/- mice, incubation with Klotho induced an increase in ACh-induced relaxation (Figure
5B), which was significant only at 3.0*10-6 M ACh.
Figure 5. Recombinant mouse Klotho effects on acetylcholine-induced vasodilation. (A) Responses from rings from Klotho-/- mice (N = 3) treated with 0.4 nM Klotho (N = 5 rings) or PBS (N = 4 rings) to acetylcholine (mean ±
SEM). (B) Responses from rings from 90-week-old Klotho+/- mice (N = 4) treated with 0.4 nM Klotho (N = 8 rings)
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Discussion
The goal of this study was to re-visit the relationship between Klotho and vascular function and we found that Klotho-/- mice have marked endothelial dysfunction. Surprisingly, these
mice were more sensitive to phenylephrine. Also surprisingly, we could not observe endothelial dysfunction in Klotho+/- mice. Finally, this study provides indications that soluble
Klotho may rapidly improve endothelial function in Klotho-/- and Klotho+/- aortic rings.
After the serendipitous discovery of Klotho was first described in 1997, the first study on vascular function in Klotho-deficient mice followed suit in 1998 and was performed by Saito
et al. They describe that kl/+ mice (heterozygotes of the original kl/kl mice in which the Klotho
promoter is disrupted) display endothelial dysfunction (14). Also, Saito et al. investigated the response of cremaster arterioles (33 ± 3 µm in diameter) in vivo in kl/kl mice and found that relaxation of these arterioles in response to ACh was impaired. However, the aortic rings from
kl/kl mice were too calcified to dilate or contract in response to ACh. The Klotho-/- mice we
used do not develop severe vascular calcification, likely due to a combination of factors, including a genetic background more resistant to the development of vascular calcification, diet composition, and housing conditions. The lack of calcification allowed us to evaluate the vascular function in aortic rings of Klotho-/- mice for the first time. It was not necessarily a
given that large conduit arteries from Klotho-/- mice would have endothelial dysfunction
because it had been detected before in heterozygote kl/+ mice. After all, kl/+ mice develop hypertension (albeit at a later age) (17, 18), whereas kl/kl mice develop hypotension (26), likely due to the additional presence of hypovolemia (27). Our results in Klotho-/- mice further
cement that endothelial dysfunction in Klotho deficiency is likely a primary phenomenon, rather than secondary to, for instance, hypertension.
An interesting finding in this study was the increased sensitivity to PE that we observed in Klotho-/- mice. This may be in line with the increased response to norepinephrine in kl/+ rings
compared to WT rings as previously reported (16). An increased sensitivity to PE has been observed before in conduit arteries from hypertensive rats (28) (which likely also had decreased Klotho levels (29)). In this study, however, there was an increased potency rather than efficacy of PE. An increased efficacy of PE has also been observed, for instance, due to increased oxidative stress (30). Of note, PE responses in aged Klotho+/- and WT animals were
similar to the PE response in young Klotho-/- mice, indicating that perhaps sensitivity to PE
increases with age and the premature ageing syndrome seen in Klotho-/- mice is accompanied
by a prematurely increased response to PE. Evidence from rat studies appears to be in line with this hypothesis (31, 32), as well as evidence from the SAM-P8 mouse (33), another mouse model of premature ageing.
Our results disagree with the literature in an important aspect, which is that we could not identify any differences in vascular function between Klotho+/- and WT animals. Since a
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number of phenotypic aspects in this model are less severe than in other Klotho-deficient mice (like the development of vascular calcification), it is possible that Klotho+/- mice are protected
from developing endothelial dysfunction as well. Since Klotho+/- mice are generally
indistinguishable from WT littermates, save for their increased vulnerability to renal, cardiac, and other damage, it is clear that Klotho levels of approximately 50% of WT levels are typically enough to prevent the spontaneous development of ageing-related phenotypes. It is possible that the mice used in this study remained above the threshold below which endothelial dysfunction develops and that a different diet, different housing conditions or a different genetic background would have potentiated the development of endothelial dysfunction also in Klotho+/- mice.
Saito et al. also observed that vascular function in kl/+ mice is restored by parabiosis with WT littermates (14), indicating that (a) soluble factor(s) more abundant in WT mice, or WT-assisted excretion of (a) soluble factor(s) more abundant in Klotho-deficient mice is responsible for improving vascular function. Given the beneficial effect of Klotho overexpression on endothelial dysfunction in OLETF rats (15), and the occurrence of Klotho as a soluble factor in the circulation, it is tempting to speculate that soluble Klotho itself from WT mice was responsible for improving vascular function in kl/+ mice after parabiosis. However, as Klotho deficiency entails profound disturbances in a great many pathways, there are certainly other soluble factors that are plausibly of relevance. For instance, hyperphosphatemia can cause endothelial dysfunction (34, 35), as can high levels of uremic toxins (36, 37). Both phosphate and indoxyl sulfate are elevated in Klotho-deficient mice (38). The function of membrane-bound Klotho as a co-receptor to fibroblast growth factor (FGF) receptor 1c for FGF23 (39, 40)
results in high FGF23 levels in the absence of Klotho and high FGF23 levels can also cause endothelial dysfunction (41, 42). Despite these alternative reasons for endothelial dysfunction in Klotho deficiency, our data support that there could also be a direct effect of soluble Klotho, although our experiments lack the power for conclusive statements. Because aortic rings were pre-incubated with recombinant Klotho and Klotho was present during ACh stimulation, we cannot currently discriminate between an effect initiated by recombinant Klotho incubation and potential changes in protein expression, or an acute effect of the presence of Klotho during relaxation, as described by Six et al. (43). However, the divergence of the curves occurred only during stimulation with higher concentrations of ACh (at a constant Klotho concentration), which is suggestive of an effect mediated by Klotho pre-incubation. Further study of this phenomenon will be required to provide clear answers.
The nature of the interaction between Klotho and the endothelium is an important piece in many puzzles in Klotho biology. There are indications that Klotho binds directly to the endothelium (44) and it has been shown that Klotho prevents excessive calcium influx into endothelial cells by promoting internalization of calcium channel TRPC1 via a complex with Klotho and VEGFR2 (21). Furthermore, while it recently became clear that Klotho binds to lipid rafts in the cell membrane (45), it is unclear whether this also occurs in endothelial cells and there also appears to be a yet to be elucidated way for Klotho to cross the endothelium via
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transcytosis (46-48), through which it may reach target cell types in the body. Further study of the interaction between Klotho and the endothelium therefore holds great potential for vascular disease and ageing-related diseases in general.
In conclusion, we can confirm that Klotho deficiency is accompanied by endothelial dysfunction. Furthermore, Klotho-/- mice display an exaggerated response to phenylephrine,
which is potentially ageing-related, and there may be direct protective effects of soluble Klotho on endothelial dysfunction. A therapeutic approach of preventing endothelial dysfunction using Klotho-based treatment merits further investigation.
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Acknowledgments
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