University of Groningen
Klotho in vascular biology
Mencke, Rik
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Publication date: 2018
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Mencke, R. (2018). Klotho in vascular biology. Rijksuniversiteit Groningen.
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Chapter 5
Assessment of vascular Klotho expression
and functionality
R. Mencke Z. Zhuge M. Burmakin G. Harms M.G. Vervloet M. Carlström H. Olauson J.L. Hillebrands on behalf of the NIGRAM Consortium166
Abstract
The renal anti-ageing protein Klotho is being increasingly viewed as a possible link between chronic kidney disease (CKD) and its cardiovascular complications. Klotho is primarily expressed in the kidney, where it is proteolytically cleaved and circulates as soluble Klotho, having beneficial effects on the vasculature. While renal Klotho and soluble Klotho are potentially useful in targeting the vasculature, endogenous Klotho expression in the vasculature is a controversial topic. Various studies conclude that Klotho is not expressed while a multitude of other studies reports high smooth muscle Klotho expression.
In furtherance of resolving this issue, we investigated vascular Klotho expression in mouse and human using Western blotting, immunohistochemistry, PCR, and RNA in situ hybridization. We performed various controls using Klotho-/- mice, Six2-Cre/Klothoflox/flox mice, and
SM22α-Cre/Klothoflox/flox mice and further investigated whether SM22α-Cre/Klothoflox/flox mice have a
vascular phenotype as reflected by impaired vascular function.
We detected a 116 kDa band on Western blot in cultured vascular smooth muscle cells, as well as a vascular staining pattern in tissues, using anti-Klotho antibody Ab69208. This staining pattern did not overlap with the known renal Klotho expression pattern. The Ab69208 staining pattern was present in both Klotho-/- mice and SM22α-Cre/Klothoflox/flox mice, indicating that
it is not specific for Klotho. We corroborate, however, that Klotho is expressed at an extremely low level in the vasculature as detected by RT-PCR, which is further underlined by the finding that SM22α-Cre/Klothoflox/flox mice have a phenotype including endothelial dysfunction and
impaired contractility.
In conclusion, our data indicate that detection of Klotho as a 116 kDa band or as a vascular staining pattern is not related to Klotho, but there appears to be a role for the very low Klotho expression level in regulating endothelial function and smooth muscle cell contractility.
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Introduction
There has been a growing interest in the relationship between the pathophysiology of chronic kidney disease (CKD), the occurrence of cardiovascular disease (CVD) in CKD patients, and the possibility that the anti-ageing protein Klotho could be a factor relevant to both. Klotho is a renal transmembrane protein expressed primarily in the distal convoluted tubule (1), where it functions as an obligate co-factor to fibroblast growth factor (FGF) receptor 1c facilitating FGF23 signaling (2, 3) and where it is cleaved off and released as soluble Klotho in blood and urine (4, 5). Deficiency of Klotho in mice causes a syndrome resembling human ageing (1), while Klotho overexpression extends lifespan (6) and protects against various ageing-related diseases (7-16). The combination of findings that CKD is an acquired state of Klotho deficiency (9, 17, 18), Klotho deficiency itself induces a mild form of CKD (9, 19), deficiency of Klotho especially affects the heart and the vasculature (1, 7, 9, 20), and Klotho treatment is beneficial to both experimental CKD and its cardiovascular complications (7, 9, 21), strengthens the hypothesis that Klotho constitutes an important link between CKD and CVD. One promising area of research therefore explores the targeting of Klotho as a treatment for the vasculature, which is afflicted with vascular calcification (1), endothelial dysfunction (22, 23), arterial stiffening (24-26), and hypertension (19, 27) in both murine Klotho deficiency and CKD. Which form of Klotho to target, however, is an open-ended question. Soluble Klotho has been shown to be able to ameliorate Klotho deficiency-induced vascular calcification (28). The alternatively spliced transcript long thought to encode a putative “secreted Klotho”, however, is not actively translated to a protein (29), but therapeutic strategies focused on the KL1 internal repeat that comprises most of the putative “secreted Klotho” are likely viable (30-32). Finally, membrane-bound vascular Klotho expression is a matter of great debate. Stimulating Klotho expression systemically is expected to affect the vasculature by a potentially higher level of soluble Klotho that is derived from the kidney. Whether Klotho is expressed in the vasculature itself and whether stimulating endogenous vascular Klotho expression would therefore be an option to be explored, is currently highly controversial. We and others have previously shown that membrane-bound Klotho protein could not be detected in human or mouse arteries and that, lacking Klotho as an obligate co-receptor, the vasculature does not partake in down-stream FGF23 signaling like the kidney does (33, 34). Our data also indicate, though, that Klotho mRNA is expressed at an extremely low level in the vasculature. This finding is widely echoed in recent literature, but interestingly, many authors also report the presence of high levels of Klotho protein in arteries and smooth muscle cells (35-37). Therefore, in this study, we investigated arterial Klotho expression in furtherance of resolving this issue.
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Materials and Methods
Human tissue
Healthy renal artery pieces from kidney donor (leftover from surgery; N = 6) and kidney graft recipient iliac arteries (leftover during creation of the end-to-side anastomosis; N = 6) were collected at the VU University Medical Center, after approval by the Institutional Review Board and after informed consent was given. Renal cell carcinoma-adjacent healthy renal tissue removed during nephrectomy was used as control tissue. Use of human material was conform the Declaration of Helsinki.
(Immuno)histochemistry and immunofluorescence
For immunohistochemistry and immunofluorescence, we used 4 µm thick cryosections, which were dried, fixed in acetone at RT for 10 minutes, followed by incubation with anti-Klotho antibodies Ab69208 (1:100; Abcam, UK) or KM2076 (1:20; TransGenic Inc., Japan), or with E29 (for Epithelial Membrane Antigen, Dako, Denmark) or 1A4 (for α-Smooth Muscle Actin, Dako) for 1 hour. Endogenous peroxidase was inactivated in 0.075% H2O2 in PBS for 30 minutes.
Polyclonal secondary and tertiary antibodies were applied as appropriate (rabbit anti-rat-HRP (P0450, Dako), goat anti-rabbit-HRP (P0448, Dako), rabbit anti-goat-HRP (P0449, Dako), donkey anti-rabbit-Alexa Fluor 488 (Invitrogen), goat anti-mouse IgG2a-Alexa Fluor 555 (Invitrogen)). As a chromogen, 3-amino-9-ethyl carbazole (AEC) was used (with 0.03% H2O2 in
0.02% AEC/50 mM acetate) for 15 minutes. Nuclei were counterstained with hematoxylin or DAPI. Hematoxylin-eosin stainings, periodic acid-Schiff stainings, and Von Kossa stainings were performed according to routine protocols on FFPE sections.
Cell culture
Human aortic smooth muscle cells (HASMCs, ScienCell, USA) were cultured in Smooth Muscle Cell Medium with 2% Fetal Calf Serum, 1% penicillin/streptomycin, and 1% Smooth Muscle Cell Growth Supplement (all from ScienCell), at 37 °C and 5% CO2. Human kidney epithelial cell line HK-2 was maintained in DMEM with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin (all from Lonza, USA). For cytofluorescence, HASMCs were cultured on glass coverslips, fixed in 2% paraformaldehyde, and stained according to the protocols described above, but with Tyramide-TRITC Signal Amplification (Perkin Elmer, USA).
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Western blotting
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and equal amounts of protein were enzymatically de-glycosylated in loading buffer using Endoglycosidase F (a kind gift from Prof. Joost Hoenderop, Randboud University Nijmegen, The Netherlands) for 1 hour at 37 °C. Proteins were separated on 8% polyacrylamide SDS-PAGE gels and blotted onto nitrocellulose membranes. After blocking with 5% non-fat dried milk/TBST, blots were incubated with Ab69208 or KM2076 1:1000 overnight, followed by appropriate secondary antibodies, and detection with Super Signal West Femto Chemiluminescence (Thermy Scientific, USA) on a ChemiDoc MP Imaging System (Bio-Rad, USA).
PCR
RNA was extracted using the TRIzol (Thermo Fisher Scientific) method with chloroform and 2-propanol. Synthesis of cDNA was performed using SuperScript II (Thermo Fisher Scientific) and random hexamer primers. For PCR 20 ng of cDNA input was used, for 40 cycles of 30 s at 94 °C, 30 s at 59 °C and 30 s at 72 °C. Primer sequences are as follows: exon 1 forward (5’-ACTACCGCTTCTCCATCTCG-3’) with exon 2 reverse (5’-TCAAGGTCAATCCAGGAAAG-3’), exon 2 forward CCACAGCATCAAAGAATGTC-3’) with exon 3 reverse (5’-CCACAGCATCAAAGAATGTC-3’), exon 3 forward (5’-CTAAGCCAGGACAAGATG-3’) with exon 4 reverse (5’-TCAGGTCGGTAAACTGAG-3’), and exon 4 forward (5’-CTCCAGGAAATGCACGTTAC-3’) with exon 5 reverse (5’-AAAGCCAGTAAAGACTTTCG-(5’-CTCCAGGAAATGCACGTTAC-3’), followed by resolving on a 2% agarose gel.
RNA in situ hybridization
Formalin-fixed, paraffin-embedded kidney sections from WT and SM22α-Cre/Klothoflox/flox
mice were baked at 60 °C for 1 hour, followed by antigen retrieval and RNA-FISH or RNA-BRISH according to manufacturer instructions (ACDBio, Italy). The probes we used were directed against the mouse Klotho gene (422081) and the human Klotho gene (422041) with the RNAscope 2.5 HD Reagent kit or the RNAscope Multiplex Fluorescent kit v2.
Animal experiments
Klotho+/- mice were provided by Prof. Joost Hoenderop (Radboud University Medical Center,
Nijmegen, The Netherlands). Breeding at the University Medical Center Groningen Central Animal Facility produced WT, Klotho+/-, and Klotho-/- mice. Tissue from Six2-Cre/Klothoflox/flox
mice was used (38) and SM22α-Cre/Klothoflox/flox mice were generated as described previously
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the Klotho gene. All animal experiments were conform the NIH Guide for the Use and Care of Laboratory Animals. For vascular function measurements, male and female SM22α-Cre/Klothoflox/flox and WT mice (N = 4 per group) 8-12 weeks of age with an average weight of
25g mice were kept under standard conditions with free access to standard pellet chow and tap water. The mice were sacrificed by cervical dislocation. The whole intestine with attached mesenterical tissues were harvested and mesenteric arteries were dissected (215+/-25 um in diameter, 2 mm in length) and mounted in a myograph chamber (Model 620 M, Danish Myo Technology, Denmark), with physiological salt solution (PSS; composition in mM: 119 NaCl, 4.7 KCl, 1.6 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.1 NaHCO3, 5.5 glucose, 0.026 EDTA). Isometric tension was recorded with a Powerlab system (Powerlab 4/30, AD Instruments, Australia). After being mounted, the vessels were equilibrated for 30 min in PSS bubbling with carbogen (95% O2; 5% CO2) at 37 degree, pH 7.4. Resting tension of the arteries was set as described previously, which is in accordance with Mulvany’s normalization procedure. The vascular viability was verified by a contractile response to potassium chloride (KCl, 100 mM). After washing, the mesenteric arteries were pre-contracted with increasing concentrations of Phenylephrine (PE, 0.0001–10 μM) to reach approximately 80% of KCl-induced contraction. After reaching a stable plateau phase, the endothelium-dependent relaxation was induced by a cumulative concentration of acetylcholine (ACh, 0.001–100 μM). For the endothelium-independent relaxation, a cumulative concentration-dependent response for sodium nitroprusside (SNP, 0.001–100μM) was induced. Between each concentration-response curve, the arteries were washed three times with PSS solution and left to stabilize for 30 minutes.
Statistical analysis
Data are presented as mean ± SEM. Differences between groups are compared using Student’s
t test or the Mann-Whitney U test, depending on the normality of the distribution, which was
tested using the Kruskal-Wallis test. A p value < 0.05 was considered significant and statistical analyses were performed using GraphPad Prism version 5 (Graphpad Inc, USA).
Results
Ab69208 detects a 116 kDa band and displays a vascular staining pattern
We first attempted to find an anti-Klotho antibody that detects a 116 kDa protein, since this is the size of the protein detected by anti-Klotho antibodies in arteries and smooth muscle cells (SMCs). Ab69208 indeed displayed immunoreactivity in human SMCs at 116 kDa (Figure 1A). Additionally, established anti-Klotho antibody KM2076 detected Klotho in renal HK-2 cells at 130 kDa (HK2 –endoF), which exhibited a mobility shift to around 116 kDa by enzymatic
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glycosylation (HK2 +endoF). No such mobility shift occurred with the SMC band, allowing for the possibility that the 116 kDa band constitutes an non-glycosylated Klotho protein. Assessing the staining pattern of Ab69208, we found that Ab69208 is highly reactive with arterial SMCs in both healthy kidney donor renal arteries and in CKD kidney graft recipient iliac arteries (Figure 1B, C). Additionally, cultured human aortic SMCs stain positive with Ab69208 (Figure 1D). Of note, while KM2076 detects Klotho protein in distal tubules and not in arteries (Figure 1E, F), Ab69208 binds to arteries and not to distal tubules (Figure 1G, H).
Figure 1. Ab69208 is immunoreactive with a vascular epitope. (A) Western blot using anti-Klotho antibodies
Ab69208 on human aortic smooth muscle cells (with and without endoglycosidase F treatment) and KM2076 on human kidney (HK-)2 cells (with and without endoglycosidase F treatment). Note that the vascular band detected with Ab69208 is about 116 kDa in size and does not display a mobility shift after endoglycosidase F treatment and that the renal Klotho as detected with KM2076 is 130 kDa in size and exhibits a mobility shift after enzymatic de-glycosylation. (B) Ab69208 displays a smooth muscle cell staining pattern in healthy donor renal arteries.
Original magnification: 400×. (C) Ab69208 displays a smooth muscle cell staining pattern in kidney graft recipient
iliac arteries. Original magnification: 400×. (D) Cultured smooth muscle cells stain positive with Ab69208. Original
magnification: 400× (inset 630×). (E) KM2076 detects Klotho in distal tubules in human kidney but not in
intrarenal arteries. Original magnification: 400×. (F) Conjugate control for (E). Original magnification: 400×. (G)
Ab69208 detects smooth muscle cells in human kidney, but no distal tubules. Original magnification: 400×. (I) Conjugate control for (G). Original magnifications: 400×.
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Ab69208 is not immunoreactive with tubules
To further determine the Ab69208 staining pattern, we performed double stainings for Ab69208 and epithelial membrane antigen (EMA), a distal tubular marker, or α-smooth muscle actin (α-SMA), a smooth muscle cell marker. We found that the Ab69208 epitope was mutually exclusive with EMA (Figure 2A-F), whereas it showed full co-localization with α-SMA (Figure 2G-L). If Ab69208 detects a smaller Klotho protein, its expression pattern would be specifically regulated in different cell types.
Figure 2. The Ab69208 staining pattern is a smooth muscle cell staining pattern without overlap with a distal tubule staining pattern. (A) Epithelial membrane antigen (EMA) staining of human kidney. (B) Ab69208 staining
of human kidney. (C) Overlay, indicating no co-localization of EMA and Ab69208, with neither antibody staining
the glomerulus. (D) Epithelial membrane antigen (EMA) staining of human kidney. (E) Ab69208 staining of human
kidney including a larger artery. (F) Overlay, indicating no co-localization of EMA and Ab69208. (G) α-Smooth
muscle actin staining of human kidney. (H) Ab69208 staining of human kidney. (I) Overlay, indicating full
co-localization of α-SMA and Ab69208 in arterioles. (J) α-Smooth muscle actin staining of human kidney, including a
larger artery. (K) Ab69208 staining of human kidney, including a larger artery. (L) Overlay, indicating full
co-localization of α-SMA and Ab69208 in arteries. Glomeruli are indicated with g. Arterioles are indicated with open arrows. The media of arteries is indicated with m. Original magnifications are 400×.
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Ab69208 is immunoreactive with Klotho knockout tissue
To assess whether the protein detected by Ab69208 could be one originating from the Klotho gene, we used various Klotho knockout strains. In WT mouse kidneys, KM2076 detected Klotho in distal tubules (Figure 3A) and not in Klotho-/- mouse distal tubules (Figure 3B), as
expected. However, the vascular staining pattern detected by Ab69208 in WT mice (Figure 3C) was still present in Klotho-/- mice, indicating that it does not detect a Klotho protein. To further
substantiate this finding, we used kidney-specific (Six2-Cre) and artery-specific (SM22α-Cre) Klotho knockout mice (34, 38). Six2-Cre/Klothoflox/flox mice did not display tubular Klotho
staining with KM2076 (Figure 3F), which was present in their WT littermates (Figure 3E), and Ab69208 vascular staining was similar in mice from both genotypes (Figure G, H). SM22α-Cre/Klothoflox/flox mice also displayed arterial staining with Ab69208 (Figure 3L) like their WT
littermates (Figure 3K) while renal Klotho expression as
Figure 3. Staining patterns of anti-Klotho antibodies in Klotho knockout mice. (A) KM2076 detects tubules in
WT mice. (B) KM2076 does not detect tubules in Klotho-/- mice. (C) Ab69208 detects arteries in WT mice. (D) Ab69208 detects arteries in Klotho-/- mice. (E) KM2076 detects tubules in WT mice. (F) KM2076 does not detect tubules in Six2-Cre/Klothoflox/flox mice with a kidney-specific Klotho deletion. (G) Ab69208 detects arteries in WT mice. (H) Ab69208 detects arteries in Six2-Cre/Klothoflox/flox mice with a kidney-specific Klotho deletion. (I) KM2076 detects tubules in WT mice. (J) KM2076 detects tubules in SM22α-Cre/Klothoflox/flox mice with an artery-specific Klotho deletion. (K) Ab69208 detects arteries in WT mice. (L) Ab69208 detects arteries in
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detected with KM2076 was also similar (Figure 3I, J). Together, these data from different and independent strains of Klotho knockout mice indicate that vascular staining detected by Ab69208 does not concern a Klotho protein (be it glycosylated or non-glycosylated).
Klotho mRNA is expressed at an extremely low level in arteries
Many studies detail that Klotho is expressed at a very low level in arteries and in cultured SMCs. We also previously (33) and in this study detect Klotho mRNA expression in SMCs (Figure 4A). However, to reliably detect Klotho mRNA expression, a higher input of cDNA (20 ng) was required for a PCR run of 40 cycles and even then detection depends on the part of the transcript and primers. Note the lack of a band after using primers spanning the exon 1-2 boundary, illustrating that the level of Klotho mRNA is low to the extent that it is often rendered undetectable. Given the sensitivity of PCR as a detection method, we also wanted to use RNA in situ hybridization as a method of providing information about Klotho mRNA
expression in a morphological context. Klotho mRNA was readily and similarly detected in distal (and to a lesser extent, proximal) tubules in kidneys from both WT and SM22α- Cre/Klothoflox/flox mice (Figure 4B-E). In arteries, however, Klotho mRNA could not be detected
regardless of genotype (Figure 4D-I). In human kidney, Klotho mRNA was highly expressed in distal tubules, expressed at a very low level in proximal tubules, and not detectable in arteries (Supplemental Figure 1).
Phenotyping of SM22α-Cre/Klothoflox/flox mice
Recent studies have found distinct and functional effects for Klotho endogenously expressed at a very low level in bone cells (in Dmp1-Cre/Klothoflox/flox (39) and Prx1-Cre/Klothoflox/flox mice
(40)) and in proximal tubular epithelium (in Scl34a1-Cre/Klothoflox/flox, PEPCK-Cre/Klothoflox/flox,
and Kap-Cre/Klothoflox/flox mice (41)). To assess whether this could also be the case for the
vasculature, we investigated the aortas of SM22α-Cre/Klothoflox/flox mice. As reported before
(34), compared to WT littermates, artery-specific Klotho knockout aortas displayed normal morphology (Figure 5A, B, D, E) and did not display vascular calcification (Figure 5C, F), a prominent feature in systemic Klotho deficiency.
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Figure 4. Investigation of Klotho mRNA expression. (A) RT-PCR for Klotho in human kidney and human smooth
muscle cells, for 40 cycles and with 20 ng of input, yielding a Klotho band that is usually, but not always detected, which is illustrative of the low expression pattern. (B) RNA in situ hybridization for Klotho mRNA in WT mouse
kidney. (C) RNA in situ hybridization for Klotho mRNA in WT mouse kidney. (D) RNA in situ hybridization for Klotho
mRNA in SM22α-Cre/Klothoflox/flox mouse kidney. (E) RNA in situ hybridization for Klotho mRNA in SM22α-Cre/Klothoflox/flox mouse kidney. Note the artery and that Klotho mRNA expression is similar when comparing both genotypes. (F) RNA in situ hybridization for Klotho mRNA in WT mouse artery. (G) RNA in situ hybridization for
Klotho mRNA in WT mouse artery. (H) RNA in situ hybridization for Klotho mRNA in SM22α-Cre/Klothoflox/flox mouse artery. (I) RNA in situ hybridization for Klotho mRNA in SM22α-Cre/Klothoflox/flox mouse artery. Note that Klotho mRNA is not detected in arteries from either genotype. Original magnif×ication: 630×.
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Artery-specific Klotho knockout mice have endothelial dysfunction
To assess whether artery-specific Klotho knockout mice display a local vascular phenotype, we performed vascular function measurements. In SM22α-Cre/Klothoflox/flox mesenteric artery
rings, endothelium-dependent relaxation was significantly impaired (Figure 6A), whereas endothelium-independent relaxation was similar (Figure 6B). These results indicate that Klotho-deficient SMCs are capable of normal relaxation, but not when dependent on endothelium-derived NO, suggesting that cross-talk that at least includes communication from smooth muscle to the endothelium is impaired in the absence of a low level of endogenous SMC Klotho.
Figure 5. Phenotyping of the SM22α-Cre/Klothoflox/flox mouse aorta. (A) Hematoxylin-eosin staining of WT mouse
aorta. (B) Periodic acid-Schiff staining of WT mouse aorta. (C) Von Kossa staining of WT mouse aorta. (D)
Hematoxylin-eosin staining of SM22α-Cre/Klothoflox/flox mouse aorta. (E) Periodic acid-Schiff staining of SM22α-Cre/Klothoflox/flox mouse aorta. (F) Von Kossa staining of SM22α-Cre/Klothoflox/flox mouse aorta. No morphological abnormalities or calcifications were noted. Original magnification: 400×.
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Artery-specific Klotho knockout mice display impaired arterial contractility
In addition to the relaxation phenotype dependent on thendothelium, we found that artery-specific Klotho knockout mouse arterial rings exhibit impaired contractility in response to phenylepinephrine (Figure 7A), concerning an absolute difference in exerted force, rather than a relative difference compared to KPSS-induced contraction (Figure 7B). Induction of contraction by angiotensin II revealed a similar trend, which did not reach significance (Figure 7C, D).
Figure 6. Artery-specific Klotho deletion results in endothelial dysfunction. (A) Endothelium-dependent vascular
relaxation in response to acetylcholine is significantly reduced in mesenteric arteries from SM22α-Cre/Klothoflox/flox mice. (B) Endothelium-independent vascular relaxation in response to sodium nitroprusside is similar when comparing WT mice and SM22α-Cre/Klothoflox/flox mice. Plotted are means ± SEM (N = 16 rings from 4 mice, for each group). * p < 0.05.
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Discussion
The most important findings of this study are the determination that the vascular Klotho immunoreactivity of Ab69208 is non-specific and that, on the other hand, the very low expression level of Klotho does have local functions as its deficiency leads to a phenotype of endothelial dysfunction and impaired contractility.
Figure 7. Artery-specific Klotho deletion results in impaired vascular contractility. (A) Force exerted by
mesenteric artery rings from SM22α-Cre/Klothoflox/flox mice in response to phenylephrine is significantly lower compared to WT littermates. (B) Phenylephrine-induced constriction relative to KPSS-induced constriction is
similar across genotypes. (C) Force exerted by mesenteric artery rings from SM22α-Cre/Klothoflox/flox mice in response to angiotensin II tends to be lower compared to WT littermates, but the difference does not reach statistical significance. (D) Angiotensin II-induced constriction relative to KPSS-induced constriction is similar
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In recent years, many authors have addressed the question of endogenous vascular Klotho expression. With regard to Klotho protein, very discrepant findings have been reported. The first extensive study of the Klotho expression pattern was performed using a LacZ reporter system, which did not reveal appreciable vascular expression (42). Klotho expression was not detected in rat aorta using KM2076 (33, 34, 43, 44), nor with KM2119 (34) or AF1819 (25, 45-47), all antibodies known to be mostly specific for renal Klotho and to detect Klotho at 130 kDa in protein lysates using Western blotting. Other studies, however, included data on a vascular 116 kDa Klotho protein and/or staining pattern, using Ab75023, Ab69208, Ab1813373 and Ab203576 (35, 36, 48-51), unspecified anti-Klotho antibodies (37, 52-56), as well as ABIN502138 (57), and AF1819 (in mouse) (58, 59). In particular, Zhu et al. show that the alleged vascular Klotho protein is smaller than recombinant Klotho (52) and Jimbo et al. detect Klotho in rat aorta (with rat monoclonal KM2076), like Hortells et al. (60), but not in SMCs unless transfected with a Klotho construct (61). This summary suggests that the reason for the discrepant findings is likely technical in nature, since the major difference between these studies is which anti-Klotho antibody was used. Our data indicate that the vascular staining pattern and 116 kDa band are likely non-specific, because they are readily detected in both systemic and artery-specific Klotho knockout mice. Our data also render unlikely the possibilities that a Klotho protein that has undergone differential post-translational modification or that a differentially spliced variant is the underlying explanation.
Although our experiments with Ab69208 can not necessarily be extrapolated to other anti-Klotho antibodies that exhibit a vascular staining pattern, the determination that Ab69208 antibody preferentially binds to an SMC epitope with a protein mass of 116 kDa instead of to the renal 130 kDa protein does raise the bar for validation of other anti-Klotho antibodies that exhibit a similar staining pattern. The mutual exclusivity of renal Klotho and non-specific vascular staining patterns (Figures 1, 2) is a convenient characteristic of Ab69208, because other anti-Klotho antibodies (even established antibodies like KM2076 and AF1819) may generally or under certain antigen retrieval conditions detect both the normal Klotho protein and the vascular epitope, while our data indicate that the latter would still be non-specific. Additionally, the mutual exclusivity shows that the detection of a vascular Klotho staining pattern is not a matter of a greater sensitivity, rendering the low vascular protein level detectable with more suitable antibodies, because in the presence of a strong vascular staining pattern with Ab69208, the distal tubules in the kidney remained negative. We recommend that novel Klotho expression patterns be validated using knockout tissue and RNA
in situ hybridization (62).
While the data in the literature are highly discrepant on the protein level, as discussed, the data on the mRNA level have always been much more in agreement. Using PCR, Klotho is generally detected in the vasculature around the detection limit, resulting either in low expression levels (1, 33, 34, 52, 53, 55, 58, 59, 61, 63-70) or in negative data (25, 33, 44-47, 71-75), the detection limit-adjacent nature of which the RT-PCR data in Figure 4A suitably exemplify. Note that we have, on occasion, also detected the exon 1-2 junction in SMCs, as
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well as every other exon-exon junction (as seen in Figure 4A), precluding the possibility of a shorter splice variant. A better assessment of Klotho mRNA expression levels is provided by RNA in situ hybridization, for morphological and cellular context (41, 62), or by RNA-seq, for comparison to other genes (53, 74).
Combining our Klotho protein and mRNA data, we conclude that the strong vascular staining pattern and 116 kDa band are not specific for Klotho, while a very low level of Klotho mRNA is constitutively expressed in smooth muscle cells. This results presumably in a very low level of Klotho protein expression, which is too low to be detected. Its deficiency, however, results in a phenotype. Recent studies on endogenous Klotho expression have more frequently produced similar results, especially with regard to bone-specific Klotho knockout mice (39, 40) and in experiments with endothelial cells from WT mice and Klotho-deficient mice suggestive of an extremely low constitutive expression level in WT cells (76). Although the expression in proximal tubule epithelium is comparatively higher than in bone or in the vasculature, there was also for a long time a debate on whether Klotho is expressed in the proximal tubule, which has recently been detailed using RNA-ISH and the demonstration of a phenotype (41). Similarly, there is apparently a very low level of SMC Klotho expression, which affects the endothelium (causing endothelium-dependent relaxation impairment when deficient) and the smooth muscle cells themselves (causing impaired contractility). The finding that loss of SMC Klotho impairs the functionality of the endothelium, in the presence of presumably normal systemic Klotho levels (given the normal renal expression levels) on the luminal side, is highly interesting. The effect is likely either autocrine, with a loss of SMC Klotho affecting SMC-to-EC signaling mediated by other molecules, or it is a paracrine function that targets a highly polarized basolateral EC pathway, otherwise it is unlikely that the comparatively miniscule loss of SMC Klotho could not be offset by the normal Klotho levels in the circulation. We hypothesize that the impaired contractility after artery-specific Klotho deletion is a reflection of partial de-differentiation of SMCs. Relaxation, however, was not affected, except for the endothelial component.
In conclusion, we found that the vascular Klotho staining pattern corresponding to a 116 kDa protein, as is often reported, is non-specific, but that the extremely low constitutive Klotho expression level does have relevant effects, as its SMC-specific deletion leads to endothelial dysfunction and impaired SMC contractility. It is yet to be determined how this miniscule amount of Klotho affects the vasculature and by which mechanisms, but it is becoming increasingly clear that low levels of endogenous Klotho expression have important local functions in various cell types in addition to its functions as a protein present in the blood, urine, and cerebrospinal fluid.
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Acknowledgments
This study was funded by a Dutch Kidney Foundation Consortium grant [CP10.11]. NIGRAM PIs: Joost Hoenderop and René Bindels, Radboud University Medical Center, Nijmegen, The Netherlands; Marc Vervloet and Piet ter Wee, VU University Medical Center, Amsterdam, The Netherlands; Gerjan Navis, Martin de Borst, and Jan-Luuk Hillebrands, University Medical Center Groningen, Groningen, The Netherlands.
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Supplementary Figure 1. Bright-field RNA in situ hybridization for Klotho mRNA in human kidney. (A) Klotho
mRNA is expressed at a very low level in proximal tubules but expression in arteries cannot be discerned. (B)
Klotho mRNA is expressed in distal tubules, but not in arterioles. (C) Klotho mRNA is expressed in distal tubules
and at a low level in proximal tubules. Black arrows indicate very low Klotho expression. Green arrows indicate arterioles. Original magnification: 400×.
