Lim, et al. 
 
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TITLE PAGE 
 
α-KLOTHO EXPRESSISON IN HUMAN TISSUES 
Kenneth Lim1*, Arnoud Groen2*, Guerman Molostvov5, Tzongshi Lu1, Kathryn S Lilley2, 
David Snead4, Sean James4, Ian B Wilkinson3, Stephen Ting4, Li-Li Hsiao1#, Thomas F 
Hiemstra2,3,# and Daniel Zehnder5#.  
1Renal Division, Brigham and Women’s Hospital, Harvard Medical School, USA;  
2Cambridge Centre for Proteomics (CCP), University of Cambridge, 3School of Clinical 
Medicine, University of Cambridge. UK. 
4Department of Pathology, University Hospitals Coventry and Warwickshire (UHCW), 
NHS Trust, Coventry, UK 
5Division of Translational Research, Warwick Medical School, University of Warwick, UK 
 
*Co-first authors: Kenneth Lim, MD PhD and Arnoud Groen, PhD provide equal 
contribution and share first authorship. 
#Co-senior authors: Daniel Zehnder, MD PhD; Thomas Hiemstra, MRCP PhD and Li-
Li Hsiao, MD PhD contributed equally as senior authors and share senior authorship. 
#Corresponding Authors: Daniel Zehnder, MD PhD, d.zehnder@warwick.ac.uk; 
Thomas Hiemstra, tfh24@cam.ac.uk; Li-Li Hsiao, MD PhD, lhsiao@partners.org 
 
Key words: Klotho, Proteomics, Extrarenal  
 
DISCLOSURES/CONFLICT OF INTEREST: 
  Lim, et al. 
 
 2 
K.L. received a Genzyme-Sanofi Fellowship in Nephrology grant. T.F.H. is funded by 
the NIHR award to the Cambridge Biomedical Research Centre and by NIHR grant 
14/49/147. The Cambridge Aorta Study is funded by the British Heart Foundation.  
 
The authors have declared that no conflict of interest exists. 
 
Word Count: 3598 
  Lim, et al. 
 
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ABSTRACT 
Context: α-Klotho has emerged as a powerful regulator of the aging process. To-date, 
the expression profile of α-Klotho in human tissues is unknown and its existence in 
some human tissue types is subject to much controversy. Objective: This is the first 
study to characterize system-wide tissue expression of transmembrane α-Klotho in 
humans. We have employed next generation targeted proteomic analysis using Parallel 
Reaction Monitoring (PRM) in parallel with conventional antibody-based methods to 
determine the expression and spatial distribution of human α-Klotho expression in 
health. Results: The distribution of α-Klotho in human tissues from various organ 
systems, including arterial, epithelial, endocrine, reproductive and neuronal tissues was 
first identified by immunohistochemistry. Kidney tissues showed strong α-Klotho 
expression, while liver did not reveal a detectable signal. These results were next 
confirmed by western blotting of both whole tissues and primary cells. To validate our 
antibody-based results, α-Klotho expressing tissues were subjected to PRM mass 
spectrometry identifying peptides specific for the full length, transmembrane α-Klotho 
isoform. Conclusions: The data presented confirms α-Klotho expression in the kidney 
tubule and in artery, and provides evidence of α-Klotho expression across organ 
systems and cell-types that have not previously been described in humans.  
 
Abstract Word Count: 195 
 
 
  Lim, et al. 
 
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INTRODUCTION 
The identification of the novel anti-aging protein α-Klotho in 1997 (1) first 
challenged the long-held paradigm of aging as a passive, inevitable process of 
deteriorating organ function and declining health. Since α-Klotho knock-out mice 
exhibited a shortened lifespan and transgenic mice that overexpress α-Klotho live 30% 
longer (2), aging has instead emerged as a regulated and potentially modifiable 
process. α-Klotho deficiency results in a variety of features characteristic of mammalian 
aging including organ atrophy, infertility, vascular calcification, atherosclerosis, 
osteomalacia, osteoporosis, peripheral insulin sensitivity, metabolic derangements, and 
cerebral changes (1), all of which occur in “normal” aging.   
In humans, emerging data indicate that aging is also modifiable, and subject to 
regulation by complex genomic, proteomic and environmental interactions (3). 
Premature or accelerated aging occurs in a number of human genetic disorders such as 
Werner syndrome and Hutchinson-Gilford Progeria Syndrome (HGPS), conditions that 
recapitulate many or all of the features of normal aging. Further, features of aging and a 
reduced life span accompany a number of chronic disease states in humans, including 
chronic kidney disease (CKD), cancer, diabetes, HIV and inflammatory arthropathies 
(4). Only one case of a human α-Klotho mutation has been described to date, but 
polymorphisms in the α-Klotho gene (KL-VS variant) are associated with normal human 
aging (5). Given the implications of these discoveries for human health, there has been 
much interest in molecules such as α-Klotho, as potential longevity-modulating 
therapeutic targets. 
  Lim, et al. 
 
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α-Klotho has two known human isoforms: the full length protein is a 130kDa, 
1012 amino acid (AA), single pass transmembrane protein which contains a signal 
sequence, two homologous domains (denoted KL1 and KL2), a transmembrane domain 
and a short cytoplasmic tail. The second isoform (62kDa, 549AA) arises from alternative 
splicing and is a secreted soluble protein (hereafter referred to as Sα-Klotho) which 
contains only the SS domain and KL1, with the terminal 15 residues replaced by 
SQLTKPISSLTKPYH (Figure 1A)(5). Although the secreted isoform predominates and 
circulates in plasma, its function is largely unknown. In contrast, full-length α-Klotho is 
involved in aging and in phosphate homeostasis. α-Klotho functions as a co-receptor 
with the FGF receptor for the phosphatonin FGF23. Additional pleiotropic functions have 
been ascribed to tissue α-Klotho including protection against oxidative stress (6), 
inhibition of apoptosis (7) and fibrogenesis (8), promotion of angiogenesis and 
vascularization (9), vasculo-protective properties (10) (11) and regulation of stem cell 
proliferation through modulation of Wnt signaling (12), all of which may protect against 
ageing.  
α-Klotho protein expression has been found in mice, rats and humans 
predominantly in the renal distal convoluted tubular (DTC) cells (1) and to a lesser 
extent in proximal convoluted tubular epithelial cells (PTEC) (13) and parathyroid gland 
(14). We recently reported α-Klotho expression in human vasculature (11). While α-
Klotho expression has been described in other rodent tissue-types, including the 
pituitary gland, pancreas, ovary, testis, placenta, choroid plexus of the brain (1) and 
most recently in rat aorta (15), no study has to-date systematically investigated α-Klotho 
expression in humans. Expression in some tissues, particularly the cardiovascular 
  Lim, et al. 
 
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system has been controversial (16), partly due to limitations and variability of antibody-
based techniques employed to-date which may not distinguish between α-Klotho and 
Sα-Klotho. Given its implications for human health, data on the expression of α-Klotho in 
human tissues are urgently needed. 
To overcome the above limitations, we employed state-of-the-art targeted 
proteomic analysis using Parallel Reaction Monitoring (PRM) in parallel with 
conventional antibody-based methods to determine the distribution and precise nature 
of human α-Klotho expression in health.  
   
METHODS  
Tissue Sample and Preparation 
Cell lysates were obtained from commercially available primary cell cultures 
(SciencCell, USA; Human Epidermal Keratinocyte Lysate-adult (Product Code: HEKL-a, 
Catalog No: 2116), Human Prostate Epithelial Cell Lysate (Product Code: HPrEpiCL, 
Catalog No: 4416), Human Mammary Epithelial Cell Lysate (Product Code: HMEpiCL, 
Catalog No: 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (Product 
Code: HRPTEpiCL, Catalog No: 4106), Human Neuron Lysate (Product Code: HNL, 
Catalog No: 1526). Human hepatocytes (HepG2, SIGMA) were kindly gifted by Dr 
Graeme Alexander, University of Cambridge. Human aortic smooth muscle cells (HA-
SMCs; Caltag (Sciencell) catalogue number SC-6110). 
Human tissues for immunohistochemistry and tissue lysate preparation were 
obtained with local ethical approval and informed written consent from four sources: 
  Lim, et al. 
 
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Surgical specimens from the human tissue bank, Department of Pathology, University 
Hospital Coventry and Warwickshire NHS Trust, UK (Ethics approval 13\WM\0072), 
artery and kidney tissue from Warwick Medical School, UK (Ethics approval 
05/Q2802/26 and 10/H12111/36), aorta form the Department of Medicine, University of 
Cambridge (Ethics approval 05/MRE04/7), and neuronal tissue was kindly donated by 
the New York Brain Bank (NYBB) at Columbia University, New York. Human 
parathyroid whole cell lysate was obtained from Abcam, MA, USA (Catalogue Number: 
ab29792). 
 
Immunohistochemistry for α-Klotho Protein 
The rabbit polyclonal α-Klotho antibody (Catalog No. Ab69208 and Ab181373; Abcam,) 
and rabbit polyclonal isotype antibody (Catalog No. Ab27478) were used at a 
concentration of 1:100 to 1:250 for immunohistochemistry (IHC) staining and control 
experiments on formalin fixed and paraffin embedded, glass mounted sections. Please 
see supplementary methods for details of IHC protocol. 
Assessment of α-Klotho distribution along the nephron was done morphologically. 
Determination was limited to proximal tubular and distal tubular (distal tubule and 
collecting duct). Consecutive section staining with hematoxylin and eosin was also 
used.  
 
Tissue Preparation for Western Blot and Mass Spectrometry  
  Lim, et al. 
 
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Human tissues were homogenised in liquid nitrogen and resuspended in standard RIPA 
buffer supplemented with protease inhibitors cocktail (Sigma) and 1% Triton-X-100. The 
lysates were clarified by microcentrifugation at 10,000g for 10 min at 4°C and protein 
concentration assayed by Lowry assay. As control, we used recombinant human full-
length α-Klotho protein (rh-α-Klotho) (R&D Systems, 5334-KL-025).  
Western Blot for α-Klotho Protein 
Aliquots of cell lysates containing 20µg protein were separated by SDS-PAGE and 
Western blotted with anti-human α-Klotho (Catalog No. Ab69208; Abcam, MA, USA) at 
a concentration of 1:1000. 10μg of rh-α-Klotho was used as control (see supplementary 
material). The expected protein size for the full length α-Klotho protein was 116KDa. 
 
Sample Preparation for Mass Spectrometry 
All protein samples (50g) were fractionated on pre-cast SDS PAGE gels (Biorad, 456-
1083, 4-15%, 10 well) and stained with Coomassie blue stain. All samples were either 
run with an empty lane in between or on separate gels to avoid sample-to-sample 
contamination.  
In order to restrict analyses to the full-length protein, only the gel band between 
75kDa and 150kDa was resected for analysis, thus excluding Sα-Klotho (Figure 1A). Gel 
fractions were macerated with a sterile blade and subjected to in-gel digestion. Gel 
fractions were de-stained by 3 washes with 80l of 50% acetonitrile (ACN)/50mM 
ammonium hydrogen carbonate, and washed with 100% ACN. The alkylation step was 
  Lim, et al. 
 
 9 
omitted, given the absence of Cysteine residues from target α-Klotho peptides. Tryptic 
digestion was carried out overnight at 37C with 60l of trypsin (Promega, sequencing 
grade modified, V511A) in 50mM ammonium hydrogen carbonate (0.005g/l). This 
process yielded 25μl, of which 5μl (1μl for rh-α-Klotho) was subjected to nano-LC ESI-
MS/MS and Parallel Reaction Monitoring (PRM) analysis in an Orbitrap nano-ESI Q-
Exactive mass spectrometer (Thermo Scientific), coupled to a nanoLC (Dionex Ultimate 
3000 UHPLC). 
 
Data Dependent Acquisition (DDA) and Parallel Reaction Monitoring (PRM) 
A top10 DDA MS/MS analysis was performed first to assess sample quality. Samples 
were trapped on a 100μm × 2cm, C18, 5μm, 100Ȧ trapping column (Acclaim PepMap 
100) in ulPickUp Injection mode at 4 μL/min flow rate for 10 minutes. Samples were 
then loaded on a RSLC, 75μm × 50cm, nanoViper, C18, 3μm, 100Ȧ column (Acclaim, 
PepMap) retrofitted to an EASY-Spray source with a flow rate of 350nl/min (buffer A: 
HPLC H2O, 0.1% formic acid, buffer B: 100% ACN, 0.1% formic acid. 60 minutes 
gradient; 0-5 min: 5% buffer B, 5-45 min: 5->56% buffer B, 45.1-50 min: 56%->95% 
buffer B, 50.1-60 min: 5% buffer B). Peptides were transferred to the gaseous phase 
with positive ion electrospray ionization at 1.8kV. In DDA the top10 precursors were 
acquired between 400 and 1600 m/z with a 2Th (Thomson) selection window, dynamic 
exclusion of 30 seconds, normalised collision energy (NCE) of 25 and resolution of 
70,000. For PRM, precursors were targeted in a 2Th selection window around the m/z 
of interest. Precursors were fragmented in HCD mode with NCE energy dependent on 
  Lim, et al. 
 
 10 
the target peptide. MS1 was performed at a 70,000 resolution, an AGC target of 3e6 and 
a maximum C-trap fill time of 200ms; MS/MS was performed at 35,000 resolution, an 
AGC target of 5e4 and a maximum C-trap fill time of 100ms. Spectra were analysed 
using Skyline and/or MASCOT, with manual validation. A peptide was considered 
present when at least one fragment ion was detected by Skyline, in addition to the 
identification of a clear isotope pattern of the fragment ion in the raw data, at the 
expected retention time for the parent peptide.  
 
Data processing and statistical analysis  
Skyline analysis utilised .raw Thermo files. For MASCOT searches, .mgf files were 
generated from .raw Thermo files using MSconvert, uploaded in MASCOT and 
searched against a human database (Uniprot 2013) using the following settings: 
carbamidomethyl as fixed modification, methionine oxidation as variable modification; 
25 ppm peptide tolerance, 0.8 Da MS/MS tolerance; maximum of 2 missed cleavages, 
peptide selection charge of +2, +3 or +4 and selection of a decoy database. 
 
RESULTS 
Spatial distribution of α-Klotho protein in human tissues 
Consistent with previous reports (13), we confirmed expression of α-Klotho in both the 
proximal and distal tubular epithelial cells of the kidney by IHC (Figure 2A, 2B). In 
subsequent experiments, kidney tissue served as a positive control.  
  Lim, et al. 
 
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α-Klotho is expressed throughout the arterial tree 
Expression of α-Klotho was identified in smooth muscle cells throughout the arterial tree 
(Figure 2C-H). In proximal vessels, α-Klotho was present in elastic (aorta, Figure 2C) 
and muscular artery (renal, Figure 2D). Aorta α-Klotho was expressed to a similar level 
in intima and media layer (Figure 2: C1) but much stronger in vasa vasorum of the 
adventitia (Figure 2: C2). More distally, α-Klotho was also identified in smaller arteries 
within vascular beds in kidney, thyroid, prostate and testis (Figure 2E-H). We next 
sought α-Klotho in the liver, but α-Klotho could not be identified in hepatocytes (Figure 
2I).  
 
α-Klotho is present in epithelia 
We next examined α-Klotho protein expression in epithelial tissues. α-Klotho was highly 
expressed throughout the epidermis (Figure 3A) and skin appendages such as hair 
follicles and sebaceous glands (Figure 3B). In addition, epithelial cells of both the small 
bowel (jejunum, Figure 3C) and colon (Figure 3D) also demonstrated strong expression 
of α-Klotho. 
 In the male reproductive system, α-Klotho was expressed in sertoli cells (Figure 
3E, 3F) of the testis, as well as epithelial cells of the prostate gland (Figure 3G). In the 
female reproductive system, α-Klotho expression was present in mammary epithelium 
(Figure 3H), endometrium of the uterus (Figure 3I) and salpynx (Figure 3K). 
 
α-Klotho is present in endocrine tissues 
  Lim, et al. 
 
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Examination of endocrine tissue revealed α-Klotho expression in follicular epithelial cells 
of the thyroid (Figure 4A), in the insulin producing islet cells of the pancreas (Figure 4B), 
in medullary cells of the adrenal gland (Figure 4C) and testosterone-producing Leydig 
cells (Figure 3F) of the testis.  
 
α-Klotho is present in neuronal cells 
We next examined α-Klotho expression in neural tissues. Expression was found in 
neuronal cells of the cerebral cortex (Figure 4D).  In the cerebellum, α-Klotho was 
expressed primarily in purkinje cells between the molecular and granular layer (Figure 
4E). In the spinal cord, α-Klotho was present in the motor neurons in the grey matter of 
the ventral horn (Figure 4F). In the myenteric plexus, α-Klotho protein was found in 
ganglial neuronal cell bodies (Figure 4E). 
 
Analysis of α-Klotho expression by western blotting 
In order to confirm the presence and molecular weight of α-Klotho for those tissues 
where it was identified by immunohistochemistry experiments, we next performed 
western blotting on both human tissue lysates and, where available, on human primary 
cells. For these experiments, we used full-length rh-α-Klotho protein as positive control. 
Western blotting demonstrated the presence of full-length (116kDa) α-Klotho in lysates 
from human renal PTEC, HA-SMCs, neuronal cells, keratinocytes and mammary 
epithelial cells (Figure 1C). Consistent with IHC (Figure 2I), α-Klotho could not be 
identified in hepatocytes. In human tissue lysates, full-length α-Klotho was identified at 
  Lim, et al. 
 
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116kDa in kidney, renal artery, aorta, epigastric artery, cerebral cortex, spinal cord and 
cerebellum (Figure 1D). 
 
Mass spectrometry analysis of human samples 
The data described above provide strong support for the presence of full-length  
α-Klotho in the tissues shown. However, since these data are antibody-dependent, we 
next sought to corroborate these findings using a targeted proteomics approach, PRM. 
PRM is able to robustly and reproducibly identify a peptide signature for a protein of 
interest with precision and specificity and, importantly, can yield isoform-specific data. 
This methodology is therefore able to distinguish Sα-Klotho from α-Klotho. To further 
mitigate against the potential for contamination with Sα-Klotho, samples were first 
fractionated by 1 dimensional (1D) gel electrophoresis. Only gel fragments between 75 
and 150kDa were excised for analysis, thus excluding Sα-Klotho isoform (62kDa). Full-
length transmembrane rh-α-Klotho protein served as control and allowed generation of 
a reference PRM signature.  
 First, spectra for sixteen candidate peptides unique to α-Klotho were generated 
by subjecting purified rh-α-Klotho to tandem mass spectrometry (LC-MS/MS) 
(supplementary Table 1). We next sought the presence of these peptide spectra in 
positive control samples (human kidney and PTEC lysates) by PRM, aiming to identify 
the most reliable spectra and seeking in particular peptide spectra present in α-Klotho 
but absent in Sα-Klotho. The most robust signatures were yielded by GLFYVDFLSQDK 
(α-Klotho and Sα-Klotho) and QGAWENPYTALAFAEYAR, NNFLLPYFTEDEK, 
VYYMQNYINEALK, LWITMNEPYTR (all absent from Sα-Klotho). Given that 
  Lim, et al. 
 
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GLFYVDFLSQDK is also present in Sα-Klotho, it does not distinguish between isoforms; 
in contrast, QGAWENPYTALAFAEYAR, NNFLLPYFTEDEK, VYYMQNYINEALK and 
LWITMNEPYTR are not present in Sα-Klotho and can only be identified if full-length α-
Klotho is present. We therefore restricted subsequent PRM analyses to 
GLFYVDFLSQDK and the four full-length-specific peptides described above. Figures 5A 
to 5H show representative spectra for GLFYVDFLSQDK and LWITMNEPYTR from rh-
α-Klotho, human kidney lysate and human PTEC lysate. 
 We next sought the presence of these peptide signatures in a variety of human 
tissue and cell lysates. α-Klotho extracellular domain peptide GLFYVDFLSQDK was 
present in whole kidney lysate, kidney cortex, kidney medulla, proximal tubular epithelial 
cells, parathyroid, pancreas, keratinocytes, mammary epithelial cells, prostate epithelial 
cells, neuronal cells, cerebral cortex, cerebellum and artery (aorta, renal and epigastric 
artery) (Table 1A and B). The spectrum for GLFYVDFLSQDK in human renal artery is 
shown (Figure 5D) as representative example.  
 Peptides present only in the full-length α-Klotho and absent from Sα-Klotho were 
identified in whole kidney lysate, kidney cortex, kidney medulla, proximal tubular 
epithelial cells, parathyroid, pancreas, keratinocytes, neuronal cells, cerebral cortex, 
cerebellum and artery (aorta, renal and epigastric artery), indicating the presence of full-
length α-Klotho in these tissues (Table 1A and B). Representative spectra for 
LWITMNEPYTR in human renal artery and human neuronal cells are shown in figure 
5G and 5H. Detailed peptide evidence for full-length α-Klotho in each sample is shown 
in supplementary table 2. All proteomic data have been deposited with the 
  Lim, et al. 
 
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ProteomeXchange consortium open access repository, and can be accessed at 
http://proteomecentral.proteomexchange.org. 
 
 
DISCUSSION 
 We report the identification of full-length α-Klotho protein in a wide variety of 
human tissues including the arterial tree, epithelia, endocrine and neuronal tissues. 
Previously, α-Klotho protein expression in humans had only been described in the 
kidney (13) and parathyroid glands (14), along with our own report of α-Klotho in human 
muscular artery (11). Our findings are consistent with data from rodent studies 
demonstrating α-Klotho expression in pituitary gland, pancreas, ovary, testis, placenta 
and choroid plexus of the brain (1), and suggest tissue-specific roles for α-Klotho at 
sites not involved in phosphate transport signalling.   
 The wide tissue distribution of α-Klotho is consistent with its known role in aging. 
Rodent models of α-Klotho deficiency demonstrate reduced life span with a wide range 
of tissue phenotypes including gonadal failure, arteriosclerosis, emphysema, impaired 
cognition, hearing loss, vascular calcification, cardiac hypertrophy, osteopaenia, and 
atrophy of skin, adipose tissue, thymus and skeletal muscle (1). In humans α-Klotho 
deficiency or functional variants of α-Klotho are associated with the development of 
vascular calcification (11,17), atherosclerosis (18), diabetes (19), hypertension (20), 
CKD (21), osteoporosis (22), anaemia (23) as well as various cancers such as 
hepatocellular carcinoma (24), breast cancer (25) gastric cancer (26) and renal cell 
carcinoma (27).  
  Lim, et al. 
 
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 Our data show full-length α-Klotho in elastic and muscular artery, consistent with 
our previous report (11). Other investigators have failed to demonstrate the presence of 
α-Klotho in vascular tissue (16). This apparent conflict is most likely due to differences 
in sample preparation, experimental conditions and antibodies employed, and highlights 
the difficulties inherent in antibody-based methods. Further, given the hydrophobic 
nature of full-length α-Klotho, its isolation from experimental samples is not 
straightforward. Here, data derived from PRM experiments demonstrate the presence of 
full-length α-Klotho in human artery. Given that α-Klotho deficiency results in a striking 
arterial phenotype (1) and is associated with an increased risk of coronary artery 
disease (28) and stroke (29), our data confirming the presence of α-Klotho in the human 
artery tree is of high clinical importance. 
 Expression of α-Klotho in human skin has not previously been reported. α-Klotho 
deficiency results in skin atrophy, a hallmark of the ageing process. The mechanisms by 
which α-Klotho maintains healthy skin are not known. We also identified α-Klotho in 
other epithelia including the intestine. Though it is possible that α-Klotho may similarly 
maintain intestinal epithelial health, it may equally be involved in phosphate and calcium 
transport and homeostasis. Indeed, α-Klotho may regulate expression of intestinal 
TRPV5 and TRPV6 as observed in kidney (30). Evidence for this has been shown in 
animal models examining the role of Klotho-FGF-23 in regulating phosphate 
concentration via NaPi-II transporters in intestinal epithelial cells (31) and renal distal 
tubules (32).  
 We found strong expression of α-Klotho in thyroid follicular cells suggesting that it 
may be involved in regulation of thyroid hormone production. Given that clinical and 
  Lim, et al. 
 
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subclinical hypothyroidism increases in prevalence with increasing age, α-Klotho may 
be involved in regulating synthesis of thyroid hormone (33). This may similarly be true 
for testosterone synthesis from Leydig cells, which also declines with age. Indeed, Hsu 
and colleagues have proposed the existence of a testosterone - α-Klotho feedback loop 
(34). In our study, α-Klotho expression was also found in insulin producing islet cells of 
the pancreas. This finding assumes considerable significance given emerging evidence 
that demonstrates a role for α-Klotho in regulating the insulin/IGF-1 pathway, a highly 
conserved mechanism that itself impacts on lifespan (2,35). Finally, α-Klotho was also 
found in the adrenal medulla, the site of synthesis of catecholamines, hormones that 
regulate the immediate stress response, with a significant role in the cardiovascular 
system. 
 We identified α-Klotho at various sites throughout the central nervous system 
(CNS). Whereas previous reports have demonstrated α-Klotho in the rodent brain 
choroid plexus, purkinje cells of the cerebellum (36), and studies in humans confirm the 
presence of α-Klotho in cerebrospinal fluid in health, its CNS tissue distribution has not 
previously been described in humans (37,38). The functional significance of α-Klotho in 
the CNS remains largely unknown. However, reduced cerebrospinal fluid klotho was 
found to be associated with age, and the development of Alzheimer disease, in one 
small cohort of 70 patients (38). Intriguingly, one recent report by Dubal et al 
demonstrated that the KL-VS functional variant of the α-Klotho gene was associated 
with enhanced cognition in three independent cohorts of healthy older humans (39). 
These observations suggest that α-Klotho may modulate human learning and memory, 
and may be protective against degenerative disease within the CNS.  
  Lim, et al. 
 
 18 
 Although we identified α-Klotho in a wide variety of organs and tissues, it could 
not be identified in hepatocytes. This is consistent with a recent report by Chen and 
colleagues, showing that although α-Klotho is increasingly expressed with progressive 
dedifferentiation in hepatoma cells, normal liver was negative for α-Klotho staining on 
immunohistochemistry (40). In addition, hepatocytes (including HepG2 cells used in our 
experiments) did not show α-Klotho in Western blot experiments. Although α-Klotho 
appears to be absent, an analogous system is operational in hepatocytes: the α-Klotho 
homologue, β-klotho, is expressed in hepatocytes and adipose tissue where it appears 
to have a role in glucose metabolism. Similar to α-Klotho which forms a coreceptor with 
the FGF receptor for FGF23 in phosphate signalling, β-klotho complexes with FGF 
receptors in hepatocytes to form a coreceptor for FGF19 and FGF21 (41).  
 Our study has several important strengths. First, our report is the first to 
systematically evaluate α-Klotho expression in a wide range of human tissue samples 
and cell types. Second, we have confirmed our previous observation of α-Klotho in the 
vascular tree. Third, in parallel to IHC and Western blotting, we have employed state-of-
the-art PRM to provide antibody-independent data confirming the presence of α-Klotho 
within samples. All mass spectrometry data are publically accessible. Furthermore, we 
were able to identify peptide signatures specific for isoform 1 of α-Klotho (Figure 1), 
allowing us to demonstrate with a high degree of certainty the presence of full-length 
transmembrane α-Klotho. This finding cannot be explained by tissue contamination with 
soluble exogenous α-Klotho. 
 Our findings should be interpreted against the limitations of our study. First, it has 
proven very challenging to liberate the hydrophobic full-length α-Klotho protein 
  Lim, et al. 
 
 19 
sufficiently to allow detection. Although evidence of presence is robust, the absence of 
peptides derived exclusively from full-length α-Klotho (isoform 1) should be interpreted 
with caution, for example in the case of prostate and mammary epithelial cell lines 
(Table 1A). Secondly, any study of human tissues is limited by the availability of 
experimental material, and our panel of samples and cell lines was not exhaustive.  
  
In summary, this is the first study to systematically characterise tissue expression of full-
length α-Klotho in humans. Given the influence of α-Klotho on longevity and the impact 
of its deficiency on health and aging across multiple organ systems, our data provides 
an important first step towards elucidating the role of α-Klotho in health and disease.    
 
ACKNOWLEDGEMENTS  
We are grateful to Dr M Yasmin from the School of Clinical Medicine for assistance with 
aorta samples.  
 
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