Kloto

REVIEW
published: 12 July 2022

doi: 10.3389/fragi.2022.931331
Pathobiology of the Klotho Antiaging

Protein and Therapeutic
Considerations
Gérald J. Prud’homme 1,2*, Mervé Kurt 2 and Qinghua Wang 3,4
1
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, 2Department of Laboratory
Medicine, Keenan Research Centre for Biomedical Science, Unity Health Toronto, Toronto, ON, Canada, 3Department of
Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical School, Fudan University, Shanghai, China, 4Shanghai
Yinuo Pharmaceutical Co., Ltd., Shanghai, China
The α-Klotho protein (henceforth denoted Klotho) has antiaging properties, as first
observed in mice homozygous for a hypomorphic Klotho gene (kl/kl). These mice have
a shortened lifespan, stunted growth, renal disease, hyperphosphatemia, hypercalcemia,
vascular calcification, cardiac hypertrophy, hypertension, pulmonary disease, cognitive
impairment, multi-organ atrophy and fibrosis. Overexpression of Klotho has opposite
effects, extending lifespan. In humans, Klotho levels decline with age, chronic kidney
disease, diabetes, Alzheimer’s disease and other conditions. Low Klotho levels correlate
with an increase in the death rate from all causes. Klotho acts either as an obligate
Edited by: coreceptor for fibroblast growth factor 23 (FGF23), or as a soluble pleiotropic endocrine
Cátia F. Lourenço, hormone (s-Klotho). It is mainly produced in the kidneys, but also in the brain, pancreas
University of Coimbra, Portugal
and other tissues. On renal tubular-cell membranes, it associates with FGF receptors to
Reviewed by:
Mujib Ullah, bind FGF23. Produced in bones, FGF23 regulates renal excretion of phosphate
Stanford University, United States (phosphaturic effect) and vitamin D metabolism. Lack of Klotho or FGF23 results in
Taylor Landry,
hyperphosphatemia and hypervitaminosis D. With age, human renal function often
East Carolina University, United States
deteriorates, lowering Klotho levels. This appears to promote age-related pathology.
*Correspondence:
Gérald J. Prud’homme Remarkably, Klotho inhibits four pathways that have been linked to aging in various ways:
[email protected] Transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), Wnt and NF-κB.
These can induce cellular senescence, apoptosis, inflammation, immune dysfunction,
Specialty section:
This article was submitted to fibrosis and neoplasia. Furthermore, Klotho increases cell-protective antioxidant enzymes
Molecular Mechanisms of Aging, through Nrf2 and FoxO. In accord, preclinical Klotho therapy ameliorated renal,
a section of the journal
Frontiers in Aging
cardiovascular, diabetes-related and neurodegenerative diseases, as well as cancer.
Received: 28 April 2022
s-Klotho protein injection was effective, but requires further investigation. Several drugs
Accepted: 06 June 2022 enhance circulating Klotho levels, and some cross the blood-brain barrier to potentially act
Published: 12 July 2022
in the brain. In clinical trials, increased Klotho was noted with renin-angiotensin system
Citation:
inhibitors (losartan, valsartan), a statin (fluvastatin), mTOR inhibitors (rapamycin,
Prud’homme GJ, Kurt M and Wang Q
(2022) Pathobiology of the Klotho everolimus), vitamin D and pentoxifylline. In preclinical work, antidiabetic drugs
Antiaging Protein and (metformin, GLP-1-based, GABA, PPAR-γ agonists) also enhanced Klotho. Several
Therapeutic Considerations.
Front. Aging 3:931331.
traditional medicines and/or nutraceuticals increased Klotho in rodents, including
doi: 10.3389/fragi.2022.931331 astaxanthin, curcumin, ginseng, ligustilide and resveratrol. Notably, exercise and sport
Frontiers in Aging | www.frontiersin.org 1 July 2022 | Volume 3 | Article 931331

Prud’homme et al. Pathobiology of Antiaging Klotho
activity increased Klotho. This review addresses molecular, physiological and therapeutic
aspects of Klotho.
Keywords: aging, FGF23, hyperphosphatemia, klotho, IGF-1, NF-KappaB, TGF-beta, Wnt
1 INTRODUCTION 1) FGF23-dependent phosphate, calcium and vitamin D

regulation.
Aging is associated with changes in almost all tissues and organs 2) Antioxidant and anti-inflammatory activities.
of the body, resulting eventually in debilitating and/or fatal 3) Prevention of chronic fibrosis.
conditions such as cardiovascular disease, chronic organ failure, 4) Protective effects against cardiovascular disease.
neurodegeneration and cancer. Aging occurs at varying speed in 5) Anti-cancer (tumor suppressor) activities.
different species, suggesting some poorly defined biological clock. 6) Metabolic regulatory functions relevant to diabetes.
The molecular mechanisms underlying aging are intensely studied, 7) Anti-apoptotic and anti-senescence functions; stem cell
but still not well understood. However, the pathogenesis of age- preservation.
associated diseases is likely multi-factorial (Franceschi et al., 2018). 8) Protection against neurodegenerative disease (Alzheimer’s
In humans, a limited number of genes have been clearly linked to and other).
accelerated aging as seen in progeria (a rare condition) (Coppede
2021) or, conversely, associated with longevity (Javidnia et al., 2022). In addition to these topics, we will report on several factors
The subject of this review is Klotho (kl), which is an antiaging gene, that increase Klotho, including commonly prescribed drugs,
and the corresponding protein α-Klotho (henceforth denoted recombinant proteins, gene therapies, traditional medicines,
Klotho or KL). The gene was first identified in mice in 1997 nutraceuticals, and exercise.
(Kuro-o et al., 1997). Deficiency of the protein results in a
syndrome that has several features of aging, as observed in
mutant mice with either a hypomorphic Klotho allele (Klkl/kl) 2 SITES OF KLOTHO PRODUCTION
(Kuro-o, et al., 1997) or full knockout of the Klotho gene (Kl−/−)
(Tsujikawa et al., 2003). Klotho-deficient mice exhibit stunted Klotho is primarily produced in the kidney (renal tubules), but is
growth, renal disease, hyperphosphatemia, hypercalcemia, also found in the brain (choroid plexus, CSF and neurons),
vascular calcification, cardiac hypertrophy, hypertension, organ pancreatic β cells, blood vessels and skin (Lim K, et al., 2015).
fibrosis, multi-organ atrophy, osteopenia, pulmonary disease, Recent work also documents expression in peripheral blood
cognitive impairment and short lifespan (Bian et al., 2015; Erben circulating cells (Martin-Nunez et al., 2022). It is also excreted
and Andrukhova, 2017; Kinoshita and Kawai, 2016; Kuro-o et al., into the urine. Studies in nephrectomized rodents (Hu et al.,
1997, 2010, 2019, 2021). Overexpression of the gene has the opposite 2016), and mice with kidney-specific genetic deletion of Klotho
effects, lengthening survival (Kurosu et al., 2005). (Lindberg et al., 2014), have revealed the kidney is the principal
Klotho insufficiency appears to play a role in human aging source of circulating Klotho. It is produced by the proximal and
and, specifically, in many of the diseases that are associated with distal convoluted renal tubules, perhaps more in the distal tubules
aging. Klotho expression declines with age, renal failure, diabetes (Lim K, et al., 2015). However, some authors report similar
and neurodegenerative disease. The age-related decline in serum amounts of the membrane-bound form at both locations
levels appears to be similar in men and women; and reference (Andrukhova et al., 2012; Erben and Andrukhova, 2017).
values have recently been reported (Espuch-Oliver et al., 2022). Interestingly, Klotho has different functions in the proximal
Notably, a recent study of American adults showed that low and distal tubules (Erben and Andrukhova, 2017). In the
serum Klotho levels correlate with an increased all-cause death proximal tubule, Klotho promotes a phosphaturic effect and
rate (Kresovich and Bulka, 2021). inhibits vitamin D production, whereas in the distal tubule it
Klotho can exist as a membrane-bound coreceptor for fibroblast enhances Ca2+ reabsorption. Conditional gene knockout of
growth factor 23 (FGF23) (Urakawa et al., 2006), or a soluble Klotho in the proximal tubule reproduced many of the
endocrine mediator with many functions (Figures 1, 2) (Dalton features of systemic gene knockout, confirming the importance
et al., 2017; Kuro-o, 2017). Age-related deterioration of renal of proximal tubular functions (Takeshita et al., 2017).
function results in Klotho insufficiency, and hyperphosphatemia
that contributes greatly to the aging phenotype (Kuro-o 2010, 2018,
2021). Klotho protects the kidney and promotes phosphate 3 MOLECULAR FEATURES AND
elimination (phosphaturic effect). Remarkably, independent of REGULATION
FGF23, it inhibits at least four pathways that have been linked to
aging in various ways. Klotho blocks or inhibits transforming growth 3.1 Membrane-Bound and Soluble Forms
factor β (TGF-β), insulin-like growth factor 1 (IGF-1), nuclear factor Klotho consists of a single-pass membrane protein of 1,012 aa
κB (NF-κB), and Wnt/β-catenin (Figure 3). Consequently, as will be (130 kDa), in humans, with a very short intracytoplasmic
presented in this review, Klotho exerts major effects on several segment (10 aa) (Xu and Sun 2015; Chen G, et al., 2018). The
biological processes relevant to aging and disease (Figure 4): extracellular component consists of two domains, KL1 and KL2

FIGURE 1 | Klotho structure. The membrane-bound form is single-pass, and consists of two extracellular domains (KL1 and KL2), a transmembrane segment (TM),
and a short non-signaling cytoplasmic tail (CYT). The soluble form is generated by proteolytic cleavage, usually by either ADAM10 or ADAM17 enzymes, to release the
large soluble form (s-Klotho). This is the major form found in the circulation. It can be further cleaved to generate independent KL1 and KL2 fragments, but these are either
minor forms or undetectable in the plasma.
(Figure 1). The extracellular portion can be cleaved by membrane important to note that the ADAM secretase proteins have a very
proteases, primarily ADAM10 and ADAM17 (α-secretases), and large number of substrates, and can modify many biological
this soluble α-Klotho form (s-Klotho) is released into body fluids processes. Therefore, drugs targeting these enzymes are likely
where it acts as an endocrine hormone (Xu and Sun, 2015; Dalton to lack specificity and be subject to toxicity.
et al., 2017). Additional cleavage might release smaller KL1 or The crystal structure of Klotho and its binding sites to FGF
KL2 fragments, although to our knowledge they have not been receptors (especially FGFR1c) and FGF23 have been reported (Chen
detected in the circulation. There is possibly another soluble form G, et al., 2018). No specific receptor for s-Klotho has been identified.
of Klotho; thought to be generated by alternative splicing, However, it binds to some receptors that are widely distributed, on
consisting of KL1 only and often denoted secreted Klotho. many cell types; notably FGFRs and the TGF-β receptor.
However, the sequence contains premature stop codons, and
the mRNA is degraded (Mencke, et al., 2017a). Thus, it appears 3.2 Regulation of Klotho Expression
that the vast majority, or perhaps all, soluble Klotho consists of Several factors regulate Klotho expression, as reviewed (Xu and Sun,
the KL1/KL2 form derived by proteolytic cleaving of the 2015). This may differ considerably in various tissues, and it is only
membrane-bound protein (shedding). Indeed, in rodents, partially understood. In the kidney, which is the main site of
administration of inhibitors of secretases mediating this production, various factors can alter Klotho expression under
proteolytic cleavage drastically reduce s-Klotho levels in the physiological and pathological conditions, such as circulatory
circulation (Hu et al., 2016). stress, hypertension, oxidative stress and diabetes. Klotho is
The regulation of Klotho shedding is not fully elucidated, but generally depressed in inflammatory disorders, and is a
the role of the α-secretase ADAM10 and ADAM17 is well potentially useful biomarker of inflammation (Wu and Chen, 2022).
documented, especially in renal tubular epithelial cells (Van Multiple transcription factors regulate Klotho gene expression,
Loon et al., 2015). The expression level of these enzymes including those binding at promoter sites E-box, Ap-2, PAX4,
might be increased in various ways, for example through the Sp1 and Oct-1 (Xu and Sun, 2015). PAX4 is a positive regulator.
action of insulin, growth factors and cytokines (Bzowska et al., There are PPAR-γ response elements in the 5′ flanking region of
2004; Chen CD, et al., 2007; Maretzky et al., 2008; Murphy, 2009). the gene that increase transcription. PAX4 and PPAR-γ are
In contrast, tissue inhibitors of metalloproteinases (TIMPs) block relevant to diabetes, as discussed in other sections. Vitamin D
the action of ADAM proteins (Chen CD, et al., 2007; Murphy, binds to vitamin D response elements in the promoter, to
2009). Some drugs might increase the proteolytic cleavage of upregulate Klotho transcription. In contrast, NF-κB binds to
Klotho. There are few published examples; however, ligustilide is the Klotho promoter but is inhibitory (Moreno et al., 2011;
a notable case. This natural compound increased the expression Lin et al., 2021). This might explain, at least in part, the
of ADAM10 and s-Klotho, and was protective in a mouse model lowered Klotho levels in inflammatory conditions. Notably,
of Alzheimer’s disease (see section 10.4) (Kuang et al., 2014). It is angiotensin II is a major inhibitor of Klotho transcription

4 KLOTHO INTERACTIONS WITH FGF23

AND FGFRS
The extracellular domain of Klotho binds to either FGFR1c, -3c, or
-4 (all receptor tyrosine kinases) to form a high affinity receptor for
FGF23 (Figure 2) (Chen G, et al., 2018; Urakawa et al., 2006). In
terms of nomenclature, FGF19, FGF21 and FGF23 are of often
denoted endocrine FGFs (eFGFs), because they are soluble, enter
the circulation and can act at a distance (Phan et al., 2021). FGF23
is an eFGF produced in bone that regulates renal phosphate
homeostasis, together with vitamin D and parathyroid hormone
(PTH) (Neyra et al., 2021; Rausch and Föller, 2022). The
membrane-bound and soluble forms of Klotho can both
function as coreceptors for FGF23 (Chen G, et al., 2018).
Without Klotho the affinity of FGF23 for the receptor is very
low. The activated FGFR signals through PI3K/Akt, phospholipase
Cγ (PLCγ) and Ras/MAPK/ERK. The renal physiological functions
of Klotho have been extensively reviewed (Erben and Andrukhova,
2017; Kuro-o 2017; Zou et al., 2018; Kuro-o 2019; Kuro-o 2021;
Neyra et al., 2021; Saar-Kovrov et al., 2021), and are only briefly
described here. Activation of FGFR1c regulates phosphate and
calcium exchange in the kidney. This occurs through inhibition of
the sodium-phosphate transporters NPT-2a and NPT-2c in the
proximal renal tubule, thereby reducing inorganic phosphate (Pi)
reabsorption (phosphaturic effect) (Erben, 2018). In the distal
convoluted tubule, Ca2+ resorption through TRPV5 channels is
enhanced. There is also in the distal tubule increased Na+
reabsorption, through enhanced expression of Na+:Cl−
cotransporter (NCC). Of major physiological significance,
FGF23/Klotho/FGFR1c suppresses the expression of 1α-
FIGURE 2 | The Klotho/FGFR/FGF23 signaling complex. Klotho
hydroxylase in the proximal renal tubule, which inhibits
interacts with a FGFR (frequently FGFR1c) through an extension of its KL2
domain. FGF23 fits into a groove formed by components of KL1, KL2 and the synthesis of active vitamin D, denoted 1,25(OH)2D3 (calcitriol).
FGFR. The membrane-bound and soluble Klotho forms (KL1/KL2 Among other effects, vitamin D promotes phosphate and calcium
domains) can both bind to FGFR1c and function as coreceptors (Chen G, absorption in the gut, and this will be reduced. Furthermore, in a
et al., 2018). These molecular interactions create a high affinity binding site for Klotho-independent manner, FGF23 reduces secretion of
FGF23. The activated FGFR signals through multiple pathways as illustrated,
and outlined in the text. Abbreviations: ERK, extracellular signal-regulated
parathyroid hormone (PTH), which additionally influences
kinase; FGF23, fibroblast growth factor 23; FGFR1-c, FGF receptor 1c; phosphate calcium balance (Ben-Dov et al., 2007).
MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; A deficiency of either Klotho or FGF23 in mice results in excessive
PKC, protein kinase C; PLCγ, phospholipase Cγ. 1α-hydroxylase activity, overproduction of active vitamin D, and
associated hyperphosphatemia and hypercalcemia. In Klotho-
deficient mice, hypervitaminosis D and hyperphosphatemia both
(Figure 3), and this has been attributed to the suppression of the appear to contribute to the accelerated aging phenotype (Kuro-o
positive regulator Sp1 (Zhou Q, et al., 2010). HMG-CoA 2010, 2018, 2019, 2021). However, a low phosphate diet ameliorates
reductase is also inhibitory and this can be reversed by statin disease, even though vitamin D further increases, suggesting that the
drugs, which are inhibitors of this enzyme (Narumiya et al., 2004; hyperphosphatemia is more important than vitamin D toxicity in the
Kuwahara et al., 2008) (Table 2). Statins upregulate Klotho aging syndrome. Nevertheless, ablation of vitamin D responses
mRNA, possibly by inactivating RhoA and/or diminishing the attenuated the lesions of Klotho-deficient mice (Anour et al.,
angiotensin II response (Narumiya et al., 2004). Interestingly, 2012). It has been proposed that hypersaturation of phosphate and
FGF23 is a strong negative regulator of Klotho transcription calcium ions in the blood leads to the formation of calciprotein
(Marsell et al., 2008). As a result, events that increase FGF23 particles (CPPs) (Kuro-o, 2021). The CPPs precipitate in tissues, and
production will reduce Klotho, such as chronic renal disease are thought to accelerate the pathologic changes associated with aging.
(Kuro-o, 2019). The Klotho gene is often turned off in cancer cells, The most common clinical cause of Klotho deficiency is kidney
possibly through a number of mechanisms (Rubinek and Wolf, failure, which can result from many acute or chronic conditions
2016). In particular, the Klotho promoter is GC-rich and subject (Neyra et al., 2021). Rare human mutations causing a severe
to DNA methylation, and in tumors it can be turned off by DNA deficiency of either Klotho or FGF23 are associated with
hypermethylation. Klotho is also regulated by miRNAs, and this hyperphosphatemia beginning at an early age (Ito and
appears to be of importance in cancer (Abolghasemi et al., 2019). Fukumoto, 2021). These are autosomal recessive diseases. The

FIGURE 3 | The multiple functions of Klotho. FGF23 binds to Klotho/FGFR1c receptor in the renal tubule to increase excretion of phosphate (phosphaturic effect),
regulate vitamin D metabolism, and increase calcium reabsorption. Klotho binds to the TGF-β receptor (TβRII component) to block TGF-β action. Klotho inhibits
activation of the inflammatory NF-κB pathway by preventing nuclear translocation of the active form. Klotho increases signaling in the Nrf2 pathway; inducing multiple
antioxidant enzymes and inhibiting NF-κB. Klotho blocks IGF-1 receptor signaling; this increases activation of FoxO and antioxidant responses. Klotho also blocks
activation of the Wnt pathway by binding to soluble Wnt ligands. Klotho expression is increased by PPAR-γ activation, and also by GLP-1 and GABA stimulation; but is
suppressed by angiotensin II activation of the AT1 receptor. Abbreviations: Ang II, angiotensin II; Ca2+, calcium ion; FoxO, forkhead boxprotein O; GABA, γ-aminobutyric
acid; GLP-1, glucagon-like peptide 1; IGF-1, insulin-like growth factor 1; IGFR, IGF-1 receptor; KL, membrane-bound αKlotho; NF-κB, nuclear factor κB; Nrf2, nuclear
factor-erythroid 2-related factor 2; Pi, inorganic phosphate; PPAR-γ, peroxisome proliferator-activated receptors γ; R, receptor; sKL, soluble αKlotho; TGF-β,
transforming growth factor β; TβRII, TGF-β receptor type II; Vit D, vitamin D.
subjects develop massive calcification in soft tissues, blood vessels, (Dolegowska et al., 2019; Guthrie et al., 2022). FGF21 is
and in multiple organs and anatomic sites throughout the body. involved in regulating lipid metabolism, insulin secretion and
There are bone and dental lesions. There is also evidence of glucose homeostasis (Andersen et al., 2015; Bondurant and
systemic inflammation. Treatment includes low phosphate diet, Potthoff, 2018; Dolegowska et al., 2019). FGF21 is produced in
phosphaturic drugs, anti-inflammatory drugs, and phosphate the liver, pancreatic islets, exocrine pancreas, and other tissues.
binding agents. This severe congenital deficiency of either The liver appears responsible for most of the circulating FGF21.
functional Klotho or FGF23 is not comparable to the much less The major metabolic functions of FGF21 have received
reduced Klotho expression seen in chronic diseases described in considerable attention, and it has been examined in clinical
this review. Indeed, these are not associated with the massive trials for the treatment of obesity and diabetes.
calcification observed in congenital hyperphosphatemia.
6 KLOTHO FUNCTIONS INDEPENDENT OF

5 Β-KLOTHO FGF23
The Klotho homologue denoted β-Klotho (KLB) is an obligatory 6.1 Klotho Inhibits TGF-β
coreceptor for FGF19 and FGF21, which are both eFGFs A key protective function of Klotho is the prevention of fibrosis
(Kilkenny and Rocheleau., 2016). KLB, unlike Klotho, does not (e.g., pulmonary, renal or cardiac fibrosis), which has been
appear to have endocrine functions on its own. FGF19 plays a attributed in large part to its ability to block TGF-β, although
major role in bile acid homeostasis and liver metabolism other pathways contribute (Huang Q, et al., 2020; Mencke et al.,

FIGURE 4 | Klotho deficiency associates with multiple age-related diseases. As outlined in the captions, depressed Klotho levels are linked to hyperphosphatemia,
chronic kidney diseases, multiple cardiovascular conditions, neurodegenerative diseases, several types of cancer, pulmonary fibrosis, COPD, bone disease and
diabetes (reduced β-cell mass in the pancreas). References are listed in Table 1. Abbreviations: COPD, chronic obstructive pulmonary disease; EMT, epithelial-
mesenchymal transition.
2017b). TGF-β is a pleiotropic cytokine that has been linked to serine/threonine kinase complex. TβRII phosphorylates TβRI,
aging by its promotion of cellular senescence, stem cell decline, which then phosphorylates Smad2 and Smad3 (canonical
immunologic impairment, fibrosis and several other age-related pathway). Smad2 and 3 form a complex with Smad4 (the
pathologies (Mikuła-Pietrasik et al., 2022; Pratsinis et al., 2017; common Smad), which translocates into the nucleus. There, it
Prud’homme 2007, et al., 2017b; Tominaga and Suzuki, 2019). binds to DNA and regulates transcriptional events. Multiple
This is of particular interest, because Klotho binds to the type II molecules interact with the TGF-β receptor complex, and can
TGF-β receptor (TβRII), which blocks TGF-β binding and alter the binding of TGF-β, or receptor signaling, in a positive or
receptor signaling (Doi et al., 2011) (Figure 3). In the kidneys negative fashion (Pawlak and Blobe, 2022; Prud’homme et al.,
of aging mice, Klotho levels decline, whereas TGF-β and its 2017b). For instance, either betaglycan (also denoted TβRIII),
signaling molecules increase (Oishi et al., 2021). neuropilin-1 (Nrp1) or endoglin can associate with the TGF-β
Administration of s-Klotho protein inhibits TGF-β signaling receptor, acting as coreceptors, and characteristically enhance
and protects against renal fibrosis (Doi et al., 2011). In a more TGF-β responses. Klotho also binds to this receptor, but it has the
specific way, a Klotho-derived peptide (30 amino acids) that opposite effect.
inhibits TGF-β by binding to its receptor also protected against The activation of several non-canonical (non-Smad2/3
renal fibrosis (Yuan et al., 2022). These experiments provide dependent) pathways adds more complexity (Derynck and
evidence that Klotho can suppress TGF-β activities in vivo. Budi, 2019; Aashaq et al., 2022; Baba et al., 2022). In some
Practically all cell types express TGF-β receptors, and its cells, especially endothelial cells, signaling can occur through
importance is considerable. TGF-β signaling and its regulation the alternative ALK1/Smad1,5,8 pathway. Smad4 (common
are complex (Mullen and Wrana, 2017; Derynck and Budi, 2019; Smad) then participates as it does in the Smad2/3 pathway.
Aashaq et al., 2022), and only a brief outline is provided here. The signaling TGF-β receptor can activate ERK, JNK, p38
TGF-β is secreted in latent form, and is activated by interaction MAPK, PI3K/Akt, Rho-like GTPases, NF-κB and other
with integrins and other mechanisms. Three isoforms of TGF-β pathways. Furthermore, through canonical or non-canonical
exist (TGF-β1 is the most abundant), which all bind to the same signaling, TGF-β can crosstalk with bone morphogenetic
signaling receptor (Groppe et al., 2008; Huang T, et al., 2011; proteins (BMPs), Wnt, Hedgehog, Notch, Hippo (TAZ/YAP),
Aashaq et al., 2022). This receptor consists of TβRI (also denoted JAK/STAT; as well as growth factors such as hepatocyte growth
ALK5) and TβRII. TGF-β binds to TβRII and TβRI forming a factor (HGF) and epidermal growth factor (EGF) (Luo 2017;

Mullen and Wrana, 2017; Derynck and Budi, 2019). Thus, TGF-β signaling events in the NF-κB pathways involve the activation
can impact on a vast number of biologic processes. In the immune of kinases, such as TAK1 and the NEMO complex. In the canonical
system, it is produced by regulatory T cells (Treg), and other cell pathway, in the quiescent state, the inhibitor of κB (IκB) is bound to
types, and suppresses or regulates immune responses involving the NF-κB (p50/RelA) and this prevents its activation. In response
macrophages, dendritic cells, B cells, effector T cells, NK cells and to stimulatory action, the IκB protein is phosphorylated,
neutrophils (de Streel and Lucas, 2021; Maizels 2021; ubiquitinated, and subjected to proteasomal proteolysis. This
Prud’homme 2007). Thus, it is a key regulator of the immune allows NF-κB to migrate into the nucleus and bind to DNA,
system. and induce transcription of a large number of genes. In the
The studies of Klotho inhibition of TGF-β mentioned above non-canonical pathway, RelB is bound to a protein denoted
pertain primarily to renal disease, but are likely applicable to p100. The activation of NF-κB inducing kinase (NIK) leads to
fibrosis elsewhere such as pulmonary fibrosis, and several other phosphorylation of the p100 protein, and its subsequent
age-related conditions. This would be consistent with the degradation to the p52 protein. Then, a p52/RelB complex
ubiquitous expression and multiple functions of TGF-β and its translocates into the nucleus and activates the transcription of
receptors. TGF-β is involved in many of the lesion observed in the target genes. Interestingly, there is evidence that the two
diabetes. The role of TGF-β in neoplasia is complex and context pathways interact, and might not be completely independent.
dependent. At early stages of neoplasia it acts as a tumor Major inhibitory functions of Klotho derive from its ability to
suppressor, but it aggravates cancer in the late phases, and block NF-κB signaling, as demonstrated in several studies. In this
contributes to metastatic disease (de Streel and Lucas 2021; case, Klotho appears to prevent the nuclear translocation of NF-κB,
Baba et al., 2022). Therefore, its inhibition by Klotho is although other mechanisms may also apply, as described below.
relevant to cancer therapy. For instance, Klotho suppressed TNF-α-induced activation of NF-
κB in endothelial cells (Maekawa et al., 2009). Uremia is associated
6.2 Klotho Inhibits NF-κB with circulating toxins that cause oxidative stress to endothelial
Inflammation is thought to be a major contributor to aging, in a cells and induce their senescence. These endothelial changes were
process sometimes referred to as inflammaging (de Almeida et al., attributed to NF-κB activation, and this was relieved by Klotho
2020; Fulop et al., 2021). Chronic, low-grade inflammation can (Buendia et al., 2015, 2016). The authors concluded that Klotho
lead to permanent tissue damage. For example, inflammatory prevents nuclear translocation of NF-κB, which is an essential step
changes of various types have been linked to atherosclerosis, in the activation pathway. Other investigators reported similar
chronic renal disease, diabetes-related organ injury, and findings (Yang, et al., 2012). In a diabetic cardiomyopathy model,
Alzheimer’s disease. Klotho exerts anti-inflammatory activities Klotho inhibited NF-κB in vitro and in vivo (Guo et al., 2018). This
that appear to be independent of FGF23. Importantly, Klotho suppression appeared to be due to an increase in nuclear factor-
suppresses activation of the inflammatory NF-κB pathway erythroid 2-related factor 2 (Nrf2) activation, which counteracts
(Buendia et al., 2015, 2016). This pathway plays a key role in NF-κB (see below). In diabetic db/db mice, Klotho expression was
initiating immune and/or inflammatory responses mediated by reduced in the kidney, a finding linked to increased to NF-κB
B cells, T cells, macrophages and polymorphonuclear leukocytes activation (Zhao Y, et al., 2011). This was reversed by the
(PMLs) (Haga and Okada, 2022; Roberti et al., 2022). It also application of Klotho, with a consequent reduction in
inhibits apoptosis of immune cells and other cell types, while inflammatory cytokines. Klotho was also found to inhibit NF-
promoting proliferation. It is activated by T-cell and B-cell κB activation and translocation in clonal β cells (Prud’homme
costimulatory receptors, several inflammatory cytokines, et al., 2017a). In that case, Klotho knockdown with siRNA resulted
chemokines, toll-like receptors (TLR), NOD-like receptors in spontaneous nuclear translocation of NF-κB p65. This is
(NLR), stimulator of interferon genes (STING), and other consistent with Klotho preventing the degradation of the IκB
factors that promote immunity against infectious agents protein, as suggested by others studying alveolar macrophages
(Roberti et al., 2022; Haga and Okada, 2022; Zhang T, et al., (Li L, et al., 2015). Interestingly, s-Klotho added to cultures reverses
2021). However, NF-κB is also implicated in detrimental the effects of Klotho knockdown, and prevents NF-κB activation (Li
inflammation contributing to aging, chronic inflammatory L, et al., 2015; Prud’homme et al., 2017a). The mechanisms of NF-
conditions and autoimmune diseases. NF-κB is active in many κB inhibition in these various studies may differ, and are not fully
non-immune cell types, such as endothelial cells and some elucidated (Typiak and Piwkowska, 2021). Furthermore, once NF-
epithelial cells. It contributes to vascular lesions (e.g., κB is activated and has translocated into the nucleus it can bind to
atherosclerosis and vasculitis) and, importantly, it plays a the Klotho promoter and inhibit expression, as previously noted
detrimental role in cancer (Zhang T, et al., 2021). Thus, NF- (Moreno et al., 2011; Lin et al., 2021). Thus, under strong acute or
κB can contribute the age-related diseases in many ways. chronic inflammatory conditions NF-κB activation might be
The NF-κB family includes NF-κB1 (p50), NF-κB2 (p52), RelA dominant and, as a result, Klotho expression depressed.
(p65), and RelB, and the NF-κB activation pathways have been
recently reviewed (Roberti et al., 2022; Haga and Okada, 2022;
Zhang T, et al., 2021). These proteins all have a Rel homology 6.3 Klotho Activates the Nrf2 Antioxidant
domain for sequence-specific DNA binding, as well as homo- and Pathway
hetero-dimerization. There are two routes for signaling, denoted Klotho activated Nrf2 in renal, cardiovascular and neurological
the canonical and non-canocical (alternative) pathways. Early preclinical disease models (Maltese et al., 2017; Zhu et al., 2017;

Xing et al., 2021; Xiang et al., 2022). Nrf2 is a transcription factor 2022). Wnt dysregulation results in anomalies of development,
that controls responses to oxidative stress and toxins (Dinkova- various degenerative conditions and cancer. There is a canonical
Kostova et al., 2018; Qu et al., 2020; Panda et al., 2022). Its Neh2 β-catenin dependent pathway, and a non-canonical (β-catenin
domains interact with a negative regulator denoted Kelch-like independent) pathway (Hayat et al., 2022). Non-canonical has
ECH-associated protein 1 (Keap1) (Panda et al., 2022). Keap1 two subtypes, i.e., the planar cell polarity and the Wnt/Ca2+
binds to Nrf2, promoting its ubiquitination and proteasomal pathways. In canonical signaling, multiple Wnt ligands are able to
degradation. Under the influence of oxidative stress (or other bind to the cognate receptor, denoted Frizzled, to initiate a
activators), the thiol groups on cysteine residues in Keap1 are response. In the absence of a Wnt ligand the pathway is
modified and its function is negated, resulting in the release of inactive. This is due to the constant phosphorylation of
Nrf2. Then, Nrf2 translocates into the nucleus and forms cytoplasmic β-catenin, as mediated by a protein complex,
heterodimers with other proteins, and binds to an enhancer which results in its elimination by proteasomal degradation.
sequence termed antioxidant response element (ARE). ARE is However, following the binding of a Wnt ligand to Frizzle and
involved in the expression of cellular defense genes that encode, coreceptor LRP, the degradation of β-catenin is terminated, and it
for example, several antioxidant proteins and glutathione- can migrate into the nucleus to activate target genes through
conjugated coenzyme There is also an alternative pathway of interactions with the T-cell factor/lymphoid enhancer-binding
Nrf2 regulation not requiring Keap1. Nrf2 protects against factor (TCF/LEF) transcription factors. This canonical pathway
oxidative injury and, importantly, inhibits the inflammatory regulates mainly cell proliferation, involving cyclin D1, c-myc
NF-κB pathway (Gao W, et al., 2022). The activation Nrf2 by and many other proteins; whereas the non-canonical pathways
Klotho appears to be an important factor in the protection against regulate cell polarity and motility (Hayat et al., 2022). The
renal, vascular and other diseases. proliferative aspects are particularly relevant to cancer, such as
colon cancer and hepatocellular carcinoma.
6.4 Inhibition of the IGF-1 Pathway Klotho blocks Wnt activation by binding to several Wnt
The insulin/IGF-1 receptor signaling pathway has long been ligands, including Wnt1, Wnt3, Wnt4, and Wnt5a (Liu H,
linked to aging, and it is sensitive to nutrients (Mathew et al., et al., 2007; Wang and Sun, 2009). In Klotho-deficient mice,
2017; Johnson 2018). When nutrients are abundant, the excess Wnt activation promotes cell senescence, and has a
downstream mediator denoted mechanistic target of negative impact on stem cell survival (Bian et al., 2015). In the
rapamycin (mTOR; a protein kinase) is activated, and it is kidney, Wnt overexpression is associated with fibrosis (Li X, et al.,
involved in several age-related disorders. Calorie restriction 2021), and it collaborates with TGF-β in this process. In this
(CR) can mitigate this effect, and prolong life in some species respect, Wnt can also cooperate with TGF-β to induce epithelial-
(Kapahi et al., 2017; von Frieling and Roeder, 2020). Thus, it is of mesenchymal transition (EMT), which is a precursor to fibrosis
considerable importance that Klotho inhibits the IGF-1/PI3K/ (Li X, et al., 2021). Moreover, EMT has also been linked to cancer
Akt/mTOR pathway (Kurosu et al., 2005:; Xie et al., 2013). This is stem cell (CSC) differentiation (Prud’homme, 2012;
one likely mechanism by which Klotho exerts its antiaging action, Prud’homme, 2017b). CSCs are highly tumorigenic, promote
and protection against degenerative diseases (Fung et al., 2022). metastasis and resist chemotherapy.
In this case, s-Klotho blocks insulin/IGF-1 receptor activation,
preventing downstream signaling events including
phosphorylation of insulin receptor substrates (IRS) and PI3K/ 7 DIABETES AND OBESITY RELEVANCE
Akt/mTOR signaling. The mTOR inhibitory drug rapamycin
delays age-related disease such as vascular calcification in mice Interestingly, Klotho is depleted in the islets of diabetic patients
and, interestingly, it increases Klotho expression (Zhao Y, et al., (Lin and Sun, 2015a). Several investigators have reported
2015). The insulin/IGF-1 pathway also plays a role in cancer depressed circulating Klotho levels in subjects with type 1
progression, and its inhibition by Klotho is tumor suppressive. diabetes (T1D) (Keles et al., 2016; Tarhani et al., 2020;
The insulin/IGF-1 pathway connects with antioxidant Zubkiewicz-Kucharska, 2021); and type 2 diabetes (T2D)
mechanisms through the FoxO forkhead transcription factors especially when there is advanced disease (Nie et al., 2017;
(FOXOs). Blockade of insulin/IGF-1 pathways releases inhibition Fountoulakis et al., 2018; Zhang and Liu, 2018). Circulating
of the FOXOs, resulting in their nuclear migration and the levels of s-Klotho are depressed in db/db mice (T2D)
expression of several genes encoding antioxidant enzymes, (Takenaka et al., 2011) and diabetic NOD mice (T1D)
such as manganese superoxide dismutase (Yamamoto et al., (Prud’homme et al., 2020). There is evidence that Klotho plays
2005). In accord with this, FOXO activation is depressed in an important role in glucose and lipid metabolism, as reviewed
Klotho-deficient mice, but augmented in Klotho- (Razzaque, 2012; Donate-Correa et al., 2016; Wan Q, et al., 2017;
overexpressing mice. Landry et al., 2021a). Klotho-deficient mice display islet atrophy
and reduced insulin production (Razzaque, 2012). Klotho
6.5 Inhibition of the Wnt Pathway increases β-cell expression of TRPV2 and enhances Ca2+ entry
Wnt is another major pathway blocked by Klotho (Liu H, et al., and the glucose-induced response (Lin and Sun, 2012).
2007). Wnt is a signaling cascade involved in embryogenesis, FGFR1c is expressed by β cells, and its attenuation results in
stem cell biology, cell fate specification, polarity, mitosis and diabetes and reduced numbers of β cells (Hart et al., 2000). Both
migration, as recently reviewed (Hayat et al., 2022; Rim et al., Klotho and KLB are expressed by islet cells, and this suggests

TABLE 1 | Klotho insufficiency and related pathologies.
Organ/system Disease or lesion References
Kidneys Chronic kidney disease/fibrosis, hyperphosphatemia, ischemic injury, nephrectomy, toxic injury Cheng et al. (2010)
(adriamycin streptozotocin), diabetic nephropathy, calciprotein deposition (aging) Hu et al. (2013), Hu et al. (2016), Hu et al.
(2017)
Kuro-o. (2010); 2012; Kuro-o. (2019), Kuro-o.
(2021)
Maquigussa et al. (2018)
Oishi et al. (2021)
Prud’homme et al. 2017a, 2020
Saar-Kovrov et al. (2021)
Takenaka et al. (2017)
Xing et al. (2021)
Zou et al., (2018)
Cardiovascular Arterial/aortic calcification, Atheroslerosis, Cardiomyopathy, Cardiac hypertrophy, Hypertension, Chen K, et al. (2021)
Myocardial ischemic injury/infarct Freunlich et al. (2021)
Manrique et al. (2016)
Myung et al. (2022)
Martin-Nunez et al. (2022)
Olejnik et al., 2018, 2020
Tyrenkov et al. (2021)
Wang F and Zheng, (2022)
Yang et al. (2015)
Brain Alzheimer’s disease (β-amyloid and Tau protein pathologies), hippocampal neuronal loss (Klotho Dias et al. (2021)
promotes regeneration), cognitive deficits, frailty Driscoll et al. (2021)
Cararo-Lopes et al. (2017)
Fung et al. (2022)
Hanson et al. (2021)
Kundu et al. (2022)
Laszczyk et al. (2017)
Mytych, (2022)
Nietzel et al. (2021)
Sanz et al. (2021)
Cancer Loss of Klotho’s tumor suppressor function (Klotho protects against multiple cancer types) Doi et al. (2011)
Ewendt et al. (2021)
Rubinek and Wolf, (2016)
Sachdeva et al. (2020)
Lungs Pulmonary fibrosis, chronic obstructive pulmonary disease Huang Q. et al. (2020)
Gao et al. (2015)
Bones Osteoporosis, osteomalacia from chronic kidney disease Kuro-o. (2017). 2019, 2021
Tasnim et al. (2021)
Metabolism, Pancreatic β-cell apoptosis in type 1 and 2 diabetes, glucose and lipid homeostasis, regeneration of β Keles et al. (2016)
Diabetes cells, autoimmunity and inflammation (insulitis) Landry et al., 2021a,b
Lin and Sun, 2012, 2015a, 2015b
Nie et al. (2017)
Prud’homme et al. (2017a), 2020
Razzaque. (2012); Takenata et al. (2019)
these cells can respond to FGF23 (Klotho coreceptor) and FGF21 immunosuppressive and/or anti-inflammatory effect. In
(KLB coreceptor). FGFR1c promotes signaling through several addition, recombinant Klotho improved renal disease and
key pathways for β cells (Figure 2), e.g., PI3K/Akt (promotes cell hypertension in db/db mice (Takenaka et al., 2019). The
survival), RAS-MAPK (promotes mitogenic response), PLC- authors concluded that Klotho inhibited TGF-β and tumor
PPAR-adiponectin (regulates glucose and lipid metabolism). In necrosis factor (TNF) signaling, to decrease renal fibrosis.
accord with this, Klotho administered by gene transfer in mice Systemic γ-aminobutyric acid (GABA) treatment protects
demonstrated major protective effects on β cells in T1D or T2D against type 1 diabetes (T1D) in mice (Tian et al., 2004;
models (Lin and Sun, 2015a,b). Importantly, Klotho reduced β- Soltani et al., 2011; Wan Y, et al., 2015; Wang et al., 2019). It
cell apoptosis and increased the proliferation of these cells. A key induces mouse and human β-cell replication/regeneration, while
pathologic finding in NOD mice is the development of insulitis. reducing the apoptosis of these cells (Liu W, et al., 2017, 2021;
This first appears as a peri-islet mononuclear cell infiltrate Purwana et al., 2014; Sarnobat et al., 2022; Soltani et al., 2011;
(including T cells), followed by focal invasion of immune cells Tian et al., 2013; Untereiner et al., 2019). In terms of mechanisms,
into the islets, and subsequently severe infiltration and β-cell loss. we found that GABA increases the production of Klotho, in a
Klotho gene transfer reduced insulitis, suggesting an multiple low-dose streptozotocin (STZ) model of T1D

(Prud’homme et al., 2017a). GABA therapy increased Klotho in near-normal levels. However, GABA and/or GLP-1RA directly
the pancreatic β cells, kidneys and plasma. Similarly, in the case of induced Klotho expression in pancreatic β cells. Of interest, the
human islets transplanted into immunodeficient mice, we Klotho promoter has a transcription binding site for PAX4, which
observed that oral GABA treatment increased β-cell Klotho is a transcription factor essential for the differentiation of β cells
expression and replication, and diminished apoptosis (Liu W, in the pancreas (Sosa-Pineda et al., 1997; Xu and Sun, 2015).
et al., 2021). These effects were ameliorated by co-administration GLP-1 induces the expression of PAX4 in human islets (Brun
of a dipeptidyl peptidase 4 (DPP-4) inhibitor. et al., 2008), and therefore, this may (along with other factors)
In autoimmune diabetes-prone NOD mice, the injection of induce the expression of Klotho.
s-Klotho protein increased pancreatic β-cell replication and the β- In addition to diabetes, Klotho has been found to have role in
cell mass (Prud’homme et al., 2020). It also reduced insulitis, obesity. In humans, CSF Klotho levels are depressed in obese
whereas a Klotho blocking antibody had the opposite effect. subjects (Landry et al., 2021a,b). Intracerebroventricular injection
These findings were similar to those of others who employed of Klotho in diabetes-prone mice reduced food intake,
gene transfer methods (Lin and Sun, 2015a,b). In vitro, Klotho ameliorated glucose homeostasis and reduced body weight
stimulated human β-cell survival, replication and insulin (Landry et al., 2020). This was related to FGFR signaling in
secretion (Prud’homme et al., 2017a). It also inhibited NF-κB neurons. Studies by these authors showed that Klotho acts on the
activation, which could explain the anti-apoptotic effect. Klotho arcuate nucleus of the hypothalamus (ARC) to regulate
might also protect β cells through antioxidant mechanisms that metabolism (Landry et al., 2021b). Neurons and astrocytes
can be induced, for instance, by Nrf2 and FoxO (Figure 3). Note were both targeted, and FGFR signaling through PI3K
that although GABA has only been applied in preclinical diabetes, (neurons) or ERK (astrocytes) was documented. Interestingly,
there are numerous GABAergic drugs in clinical use (Palma et al., they found no correlation between systemic and CSF
2017), to treat epilepsy and other diseases. Unlike systemically concentrations of Klotho, and the mechanism of Klotho
applied GABA that is blocked by the BBB, many of these drugs regulation in the CSF remains unclear. Circulating Klotho also
cross this barrier and this can produce multiple adverse effects. appears to play and important role in metabolism. Systemic
Other than GABA, it is unknown whether GABAergic drugs Klotho treatment in mice reduced adiposity and lipid
increase Klotho levels. accumulation in the liver; and increased lean mass and energy
Paradoxically, Klotho deficient mice (KLkl/kl) are hypoglycemic, expenditure (Rao Z, et al., 2019). With Klotho treatment,
despite having low insulin levels (Razzaque 2012). This likely quantitative PCR revealed lower expression of lipogenic genes.
results from the fact that Klotho depresses insulin sensitivity
and, therefore, the cells of Klotho-negative mice are more
responsive to insulin. This raises the question of whether 8 KLOTHO AS A TUMOR SUPPRESSOR
Klotho is an appropriate agent to treat diabetes. We hypothesize
that Klotho therapy of diabetes will be helpful when normal plasma There is considerable evidence that Klotho is a tumor suppressor
levels are restored. Interestingly, overexpression of Klotho in molecule, as reviewed elsewhere (Rubinek and Wolf, 2016;
transgenic mice produced slight insulin resistance, and extended Abolghasemi et al., 2019; Sachdeva et al., 2020; Ewendt et al.,
lifespan (Kurosu et al., 2005). In experimental T1D and T2D, as 2021). This is consistent with its capacity to inhibit pathways
outlined in this review, Klotho therapy was beneficial. Whether linked to cancer, including Wnt, IGF-1, TGF-β and NF-κB, as
humans will respond similarly is unknown. In our work, Klotho outlined previously (Figure 3; Table 1). The expression of Klotho
protein was administered at a low dose, every 48 h (Prud’homme is depressed or silenced in almost all types of cancer examined.
et al., 2020). It has a half-life of only 7 h (Hu et al., 2016), which is Klotho-positive tumors generally have a better prognosis. The
relevant to efficacy and potential adverse effects. suppression of Klotho appears to occur by the same mechanism
One key issue regarding antidiabetic drugs such as GABA and as other tumor suppressor genes. For instance, this involves DNA
GLP-1 is whether their general antidiabetic effects (Wang Q, hypermethylation at promoter sites, histone modifications and
et al., 2019; Drucker, 2018) enhance Klotho indirectly, or there is miRNAs (Rubinek and Wolf, 2016; Abolghasemi et al., 2019). In
a more direct effect on Klotho gene expression. This has not been experimental tumor models, Klotho has anti-tumor effects in vivo
extensively studied, but we hypothesize this is due to a and/or in vitro. This was demonstrated in breast cancer (Wolf
combination of mechanisms. In STZ-induced diabetes, STZ is et al., 2008; Ligumsky et al., 2015), pancreatic cancer (Abramovitz
toxic to the kidney (Cheng MF et al., 2010), not just pancreatic β et al., 2011), and other cancers including colon cancer, gastric
cells, and it reduces Klotho in the circulation and kidneys cancer, and hepatocellular carcinoma (HCC). For instance, in
(Prud’homme et al., 2017a). This was almost completely pancreatic cancer, Klotho was poorly expressed in the tumor
reversed by oral GABA administration. Studies of others have (Abramovitz et al., 2011). Overexpression of Klotho, or therapy
shown that GABA treatment protects the kidneys against tubular with s-Klotho, inhibited the growth of pancreatic cancer cells.
fibrosis and atrophy, in the subtotal nephrectomy model (Sasaki Indeed, multiple studies have shown that Klotho inhibits cancer-
et al., 2007), and ischemia-reperfusion injury (Kobuchi et al., cell proliferation, colony formation and invasion; and promotes
2009). This is consistent with the fact that renal tubular cells apoptosis and autophagy (Sachdeva et al., 2020). Similarly, in
express GABA receptors (Amenta et al., 1988; Sarang et al., 2008; vivo, forced tumor overexpression of Klotho, or systemic
Takano et al., 2014). Thus, GABA may be protecting renal tubular administration of s-Klotho, inhibited the growth of several
cells against injury, such that they continue producing Klotho at types of tumors. Importantly, this includes human tumors

TABLE 2 | Examples of current clinical drugs that increase Klotho.
Category (name) Usual Clinical applications References
Renin-angiotensin-aldosterone inhibitors (losartan, valsartan) Hypertension Janic et al. (2019)

Chronic kidney disease Karalliedde et al. (2013)
Diabetic nephropathy Li X and Zhou, (2010)
Cardiac failure Lim SC, et al. (2014)
Yoon et al. (2011)
Statins (atorvastatin, pitavastatin, simvastatin fluvastatin) Hyperlipidemia Adeli et al. (2017)
Atherosclerosis Janic et al. (2019)
Ischemic cardiovascular Kuwahara et al. (2008)
and renal disease Narumiya et al. (2004)
Yoon et al. (2012)
PPAR-y agonists (rosiglitazone, ciglitazone, pioglitazone) Diabetes Chen LJ, et al. (2014)
Hyperlipidemia Cheng et al. (2017)
Huang KC, et al. (2014)
Maquigussa et al. (2018)
Shen et al. (2018)
Zhang H, et al. (2008)
mTOR inhibitor (rapamycin, everolimus) Immunosuppression Mizusaki et al. (2019)
Transplantation Tabibzadeh, (2021)
Tataranni et al. (2011)
Zhao Y, et al. (2015)
Vitamin D Vitamin D supplementation Haussler et al. (2020)
Hypocalcemia Kuro-o, (2019)
Rickets Lerch et al. (2018)
Osteoporosis Tsujikawa et al. (2003)
GLP-1 receptor agonist (exendin-4) and DPP-4 inhibitors (linagliptin, sitagliptin, vildagliptin) Type 2 diabetes Manrique et al. (2016)
Liu W, et al. (2017); Liu et al. (2021)
Son et al. (2019)
Wang Q, et al. (2019)
Yossef et al. (2020)
Metformin Type 2 diabetes Xue et al. (2019)
Pentoxifylline Peripheral vascular disease Navarro-Gonzalez et al. (2018)
Antiplasmodial (dihydroartemisinin) Malaria Zhou et al. (2022)
Endothelin-1 receptor antagonist (Atrasentan) Diabetic nephropathy Kang and Xu, 2016
Abbreviations: DPP-4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1; mTOR, mechanistic target of rapamycin, PPAR-γ, peroxisome proliferator-activated receptor-γ.
xenografted into mice. The membrane-bound and soluble forms as neuronal aggregation of Tau protein forming neurofibrillary
of Klotho both have tumor suppressor effects. tangles (NFTs) (Thal et al., 2013). It has been proposed that these
substances are toxic to neurons. Klotho confers neuronal
resistance to oxidative and endoplasmic reticulum stress, and
9 KLOTHO AND NEURODEGENERATIVE is thought to protect against the toxicity of Aβ and NFTs (Zeldich
DISEASES et al., 2014; Fung et al., 2022). Aβ is generated from the proteolytic
cleavage of amyloid precursor protein (APP) by secretases
Low Klotho levels correlate with neurodegenerative disease and (Lichtenthaler et al., 2022). APP is a type 1 transmembrane
cognitive impairment (Vo et al., 2018; Hanson et al., 2021), as well protein, and its cleavage by an α-secretase results in the
as frailty (Veronesi et al., 2021). For example, in nursing home release (shedding) of a large extracellular segment, which does
residents, low serum Klotho was associated with poor cognition, not form amyloid. This cleavage process is accomplished
frailty, dependence, and frequent falls (Sanz et al., 2021). The role of primarily by ADAM10, and is similar to the generation of
Klotho in the brain is not well understood, but there is evidence it is s-Klotho. However, processing of APP by β-secretase and γ-
neuroprotective. In the central nervous system, Klotho is produced secretase generates small secreted Aβ peptides that form amyloid.
by the choroid plexus, and is found in the CSF. It is also expressed Interestingly, α-secretase cleavage prevents the subsequent
widely throughout the brain, predominantly in grey matter areas generation of amyloidogenic peptides (Lichtenthaler et al.,
including the hippocampus (Clinton et al., 2013; Cararo-Lopes 2022). Therefore, increasing α-secretase activity, or decreasing
et al., 2017). It is produced by both neurons and oligodendrocytes. β- and γ-secretase activity, might be of benefit.
Klotho expression in the brain is decreased in aging subjects and One potential mechanism of disease involves deficient
early Alzheimer’s disease (Fung et al., 2022). In mouse models of autophagy, which is a process that normally degrades and
Alzheimer’s disease, Klotho overexpression is beneficial. removes proteins and other substances, preventing their toxic
Morphologically, in Alzheimer’s disease, the brain is accumulation in the cell. Klotho ameliorates autophagy, and this
characterized by an excess of β-amyloid (Aβ) plaques, as well might protect against neurodegenerative conditions like

TABLE 3 | Drugs in preclinical development, supplements or other therapies that increase Klotho.
Category (name) Target diseases References
y-aminobutyric acid receptor agonist (GABA) Preclinical diabetes models, streptozotocin organ injury Liu W, et al. (2021)
Prud’homme et al. (2017a)
Son et al. (2019)
Wang Q, et al. (2019)
Recombinant protein (s-Klotho, Klotho peptide, KL1) Preclinical diabetic, metabolic, renal, vascular, Ali et al. (2020)
neurodegenerative, neoplastic and other diseases Chen TH, et al. (2013)
Doi et al. (2011)
Gupta et al. (2022)
Hu et al. (2017)
Leon et al. (2017)
Maltese et al. (2017)
Mencke et al. (2017)
Myung et al. (2022)
Oh et al. (2018)
Prud’homme et al. (2020)
Rao Z, et al. (2019)
Takenaka et al. (2018); Takenaka et al.
(2019); Takenaka et al. (2020)
Yang et al. (2015)
Yuan et al. (2022)
Gene therapy and cell therapy (Klotho gene) Preclinical, multiple diseases (alternative to recombinant Arbel Rubinstein et al. (2021)
protein therapy) Franco et al. (2021)
Lin and Sun. (2015a); Lin and Sun.
(2015b)
Ni et al. (2021)
Mencke et al. (2017)
Shin et al. (2019)
Xiang et al. (2021)
Zeng et al. (2019)
Food/diet components, supplements and traditional medicines Multiple indications, geroprotective Dehghani et al. (2019)
(astaxanthin, baicalin, cordycepin, curcumin, ginseng, ligustilide, Feng & Huang, (2022)
resveratrol, tetrahydroxystilbene glucoside) Hsu et al. (2014)
Kamel et al. (2022)
Long et al. (2018)
Li SS, et al. (2018)
Lim SW, et al. (2019)
Long et al. (2018)
Ostojic & Engeset, (2021)
Zhang P, et al. (2016)
Zhang XT, et al. (2020)
Zhou et al. (2015)
Alzheimer’s (Fung et al., 2022). Klotho also appears to contribute deficient mice demonstrate severe cognitive defects. Recently,
to oligodendrocytic myelin production, and the maintenance of intermittent fasting in mice was shown to increase Klotho
white matter integrity. There is evidence of an inflammatory expression and neurogenesis in the hippocampus (Dias et al.,
component to Alzheimer’s disease, as well as some other 2021). Intermittent fasting proved superior to caloric restriction,
neurodegenerative diseases, possibly involving NF-κB and it was associated with improvement of long-term memory.
activation (Ju wang et al., 2019). Thus, as in other sites, Klotho-deficient mice did not respond to intermittent fasting. It is
Klotho may exert beneficial anti-inflammatory and anti- possible that drug therapy can achieve similar results. For
oxidative actions in the brain. Klotho might promote instance, in a rat model of metabolic syndrome, the DPP-4
differentiation of microglia to the anti-inflammatory type inhibitor vildagliptin (increases GLP-1) augmented
(M2), instead of the inflammatory type (M1) (Fung et al., 2022). hippocampal Klotho, concurrently decreased inflammatory
The hippocampus plays a major role in memory. It is one of and apoptotic biomarkers at that site, and prevented
the few regions of the brain that sustains neurogenesis in neurodegenerative changes (Yossef et al., 2020). This improved
adulthood, and there is loss of neurons in that area in memory in treated rats. Note that most DPP-4 inhibitors,
Alzheimer’s disease (Rao YL, et al., 2022). Remarkably, recent including vildagliptin, do not cross the blood-brain barrier
work indicates that Klotho stimulates neurogenesis in the (BBB) but they enhance circulating GLP-1 (Deacon, 2019),
hippocampus. This was evident, for example, when comparing which does cross. In a rat model of STZ-induced cognitive
mice with Klotho deficiency vs. those overexpressing Klotho impairment, simvastatin increased hippocampal Klotho and
(Laszczyk et al., 2017). In accord with neuronal loss, Klotho improved cognitive function (Adeli et al., 2017). Similarly,

others found that rosiglitazone treatment improved cerebral 2021; Freunlich et al., 2021; Manrique et al., 2016; Myung et al.,
Klotho expression (Chen LJ, et al., 2014). In the latter study, 2022; Martin-Nunez et al., 2022; Olejnik et al., 2018, 2020;
intracerebroventricular infusion of recombinant Klotho was also Tyrenkov et al., 2021; Wang F and Zheng, 2022; Yang et al.,
beneficial. Alternatively, some authors have delivered Klotho by 2015. Brain: Dias et al., 2021; Driscoll et al., 2021; Cararo-Lopes
gene transfer (Table 2), such as intracerebroventricular injection et al., 2017; Fung et al., 2022; Hanson et al., 2021; Kundu et al.,
of a lentiviral vector encoding Klotho (Zeng et al., 2019). The 2022; Laszczyk et al., 2017; Mytych, 2022; Nietzel et al., 2021; Sanz
findings outlined above regarding Klotho in neurodegenerative et al., 2021. Cancer: Doi et al., 201; Ewendt et al., 2021; Rubinek
pathologies have all been obtained in rodents, and it remains to be and Wolf, 2016; Sachdeva et al., 2020. Lungs: Huang Q. et al.,
determined whether they are applicable to humans. 2020; Gao et al., 2015. Bones: Kuro-o, 2017. 2019, 2021; Tasnim
To our knowledge, Klotho-based therapy has not been applied et al., 2021; Metabolism and diabetes: Keles et al., 2016; Landry
in humans with neurodegenerative diseases. Nevertheless, et al., 2021a,b; Lin and Sun, 2012, 2015a, 2015b; Nie et al., 2017;
compared to subjects with low levels of Klotho, those with high Prud’homme et al., 2017a, 2020; Razzaque, 2012; Takenaka
levels (CSF or plasma) have superior cognitive functions, and a et al., 2019.
lower incidence of dementia (Semba et al., 2014; Shardell et al.,
2016). It has been thought that Klotho levels in the blood and CSF
are independent. Contrary to that view, some investigators have 10 APPROACHES TO KLOTHO-BASED
recently reported that serum and CSF levels correlate strongly, and THERAPY
high levels predict better cognitive function (Kundu et al., 2022).
This is of major interest for future clinical studies, especially
concerning drugs or other treatments that increase Klotho. A
10.1 Drugs in Clinical Use for Various
caveat is that the reasons for discrepancies between studies are Conditions
not clear, concerning Klotho levels in the CSF or the circulation. In view of the benefits of Klotho against aging and multiple
There has been much concern about the specificity of some anti- diseases, as reported here in preclinical models (Figure 4;
Klotho antibodies used in ELISA and other assays (Kuro-o, 2019). Table 1), it is of much interest of find applications in people.
Some authors recently examined this question in human serum In humans, it is well established that plasma Klotho levels
samples from patients with a wide range of kidney function (Neyra decrease with age and in common maladies, such as chronic
et al., 2020). This was done with a widely used commercial ELISA, renal diseases, diabetes and neurodegenerative diseases. In view of
as well as immunoprecipitation-immunoblotting (IP-IB) with a this, an obvious and safe goal would be to re-establish normal
highly specific Klotho antibody. The IP-IB Klotho results levels. Some drugs have already been mentioned for their
correlated with kidney function much better than the ELISA. potential application in specific diseases. In many of the
Freeze-thaw cycles impaired Klotho detection. These findings studies reported (Table 2 and 3) the main effect was an
suggest that relying solely on an ELISA assay could be increase in Klotho levels approaching or equal to the normal,
misleading, and sample handling is important. Another relevant rather than above normal. Based on studies in mice,
observation is the wide variability in Klotho levels between overexpression might possibly extend lifespan, but the safety is
individuals. For instance, in a 18–35 age group approximately less certain because other factors (perhaps insulin resistance)
half of individuals had low levels, comparable to aged subjects, could become important. A caveat is that practically all the
whereas the other half had high levels (Espuch-Oliver et al., 2022). information available concerning Klotho-based treatment has
In the 55–85 age group, however, levels were more consistently been obtained in rodents.
low. CSF levels are also quite variable (Kundu et al., 2022). In view A surprisingly large number of therapies increase Klotho levels
of this, results obtained from small groups should be interpreted in experimental models (Tables 2 and 3), either in the circulation
with caution. The biological significance of this variability is or specific organs. Some of the compounds involved have
unclear, and long-term studies are required. received considerable attention in the literature as
A Klotho gene variant denoted KL-VS (2 amino acid geroprotective molecules (Soo et al., 2020), but usually with
substitutions in the protein) appears to provide some scant examination of their Klotho-inducing capacity. This
protection against Alzheimer’s disease, but only in the includes several drugs that are currently in clinical use for
heterozygous state (Driscoll et al., 2021; Nietzel et al., 2021). various conditions (Table 2).
Beneficial effects were only observed in some subgroups that were
studied. The mode of action of this variant is unclear although, 10.1.1 RAAS Inhibitors
interestingly, a reduction of Tau accumulation has been reported. RAAS inhibitors are the best documented Klotho-enhancing
clinical drugs; especially losartan and valsartan that block the
angiotensin II receptor (AT1). Indeed, these are some of the few
9.1 Summary of References for Disease drugs that have been shown to enhance plasma Klotho in clinical
Relevance trials (Karalliedde et al., 2013; Lim SC et al., 2014; Janic et al.,
See also Table 1. Kidneys: Cheng et al., 2010; Kuro-o, 2010, 2019, 2019). For instance, losartan increased Klotho levels by 23% in
2021; Maquigussa et al., 2018; Oishi et al., 2021; Prud’homme diabetic patients (Lim SC et al., 2014). This is consistent with the
et al., 2017a, 2020; Saar-Kovrov et al., 2021; Takenaka et al., 2017; ability of angiotensin II to suppress the expression of Klotho
Xing et al., 2021; Zou et al., 2018. Cardiovascular: Chen K, et al., (Yoon HE, 2011). Whether Klotho augmentation contributes to

the clinical effectiveness of these drugs is unknown. Of note, these rapamycin in vitro induced the expression of Klotho. The authors
results were obtained in diabetic subjects, and it remains to be proposed that Klotho was mediating the rapamycin-related clinical
determined whether the drugs will act similarly in other diseases, alterations. In a renal transplantation clinical trial, everolimus
or healthy subjects. significantly increased Klotho serum levels (Mizusaki et al., 2019).
These investigators measured Klotho levels before transplantation
10.1.2 Statins and 1 year after. They also compared patients that were treated with
Statins are another type of frequently applied drugs that increase everolimus versus those that were not. In both cases, Klotho levels
Klotho (Table 2); however, there is limited evidence from clinical were boosted by this drug. It is of interest that renal transplantation
studies. Of major interest, the administration of a low-dose itself increased Klotho, in accord with nephrectomy decreasing
combination of fluvastatin and valsartan enhanced the Klotho, and other evidence that the kidney is the main source of
expression of Klotho and SIRT1, which have both been linked circulating Klotho (Hu et al., 2016). The increased Klotho levels in
to increased longevity (Janic et al., 2019). In contrast, mTOR and the everolimus-treated patients did not correlate significantly with
NF-κB were not increased. Statins and RAAS inhibitors are a very improvements in renal function, including serum creatinine, blood
common drug combination in medicine, and this type of urea nitrogen, eGFR, calcium and phosphorus. This study was small
treatment might have an antiaging effect. Because these drugs and other immunosuppressive agents were co-administered, which
have major effects on blood pressure, lipids, renal function, limits the interpretation. In rats with chronic renal failure, oral
cardiovascular disease and other factors, it will be difficult to rapamycin treatment also increased klotho, and this improved
establish the specific contribution of Klotho. vascular calcification (Zhao et al., 2015). The benefits were
attributed to Klotho and inhibition of mTOR based on a number
10.1.3 Pentoxifylline of pharmacological and genetic methods. Notably, rapamycin could
Pentoxifylline has been shown to increase Klotho in a clinical trial not prevent the vascular disease when Klotho expression was
(Navarro-Gonzalez et al., 2018). This drug is usually applied for suppressed with siRNA, or in Klotho knockout mice. These
the treatment of ischemic peripheral vascular disease, but there studies point to rapamycin and everolimus as effective drugs for
are other indications. In diabetic patients (type 2), serum and the enhancement of Klotho levels. These drugs, however, can have
urinary Klotho were increased, but the change in the serum levels considerable adverse effects, including depressed immunity against
was relatively modest (~6%). The patients had advanced renal infectious agents, and cell toxicity in some organs. For instance,
disease, and this may explain the low response. In clinical trials, a rapamycin is toxic to pancreatic β cells (Prud’homme et al., 2013).
major factor to consider is the health of the kidney. Renal atrophy
(typically nephrosclerosis) can be severe in patients with diabetes 10.1.6 Antidiabetic Drugs
or hypertension, and because most circulating Klotho originates Several clinical anti-diabetic drugs have been shown to increase
from the kidney this can lower levels. Thus, low Klotho levels are Klotho expression in mice, e.g., GLP-1RAs, DPP-4 inhibitors,
likely to be a reflection of renal disease, and if the renal mass is metformin and peroxisome proliferator-activated receptors γ
very low then any drug treatment might fail to increase Klotho. (PPAR-γ) agonists (Table 2). Some were mentioned previously.
Metformin, which is probably the most prescribed antidiabetic
10.1.4 Vitamin D drug for T2D, increased Klotho in the circulation, kidneys and
Vitamin D supplementation also increases Klotho, as shown in urine (Xue et al., 2019). It decreased the levels of mTOR, and this
children with chronic kidney disease (Lerch et al., 2018). Children effect was reversed by Klotho suppression. Metformin has long
with mild to moderate kidney disease had decreased serum been proposed as a geroprotective drug, although there are
Klotho levels at the beginning of the study, and this was contradictory findings (Mohammed et al., 2021). In the case of
normalized by Vitamin D treatment. In contrast, those with diabetes treatment, a caveat is that metformin increases insulin
severe renal disease had unchanged Klotho levels. This sensitivity whereas Klotho has the opposite effect.
represents the limited data available from clinical work. In PPAR-γ is a transcription factor regulating insulin sensitivity
rodents, however, there is clear evidence of Klotho and adipogenesis, and agonistic drugs find applications in diabetes
enhancement by vitamin D (Table2). To explain this and lipid disorders. PPAR-γ agonists are another class of drugs that
therapeutic effect, the complex interactions underling FGF23/ enhance Klotho expression (Table 2). There is a non-canonical
Klotho/vitamin D homeostasis must be considered, as recently PPAR-responsive element in the 5′-flanking region of the human
reviewed (Haussler et al., 2020; Neyra et al., 2021). Klotho gene (Zhang H, et al., 2008), which likely explains this
activity. These authors showed that in vitro and/or in vivo,
10.1.5 Rapamycin and Everolimus troglitazone, ciglitazone and rosiglitazone increased Klotho
Rapamycin (sirolimus) and everolimus are inhibitors of mTOR in mRNA expression. They observed increased Klotho protein in
clinical use, which have both been shown to increase Klotho the kidneys. A PPAR-γ antagonist had the opposite effects.
(Table 2). Rapamycin has long been proposed as a geroprotective
drug. Clinically, rapamycin is applied primarily as an 10.1.6.1 Summary of References for Clinical Drugs That
immunosuppressive drug to prevent transplanted organ rejection. Increase Klotho
The treatment of renal transplant recipients with rapamycin See also Table 2. Renin-angiotensin-aldosterone inhibitors: Janic
produced hypophosphatemia and insulin resistance (Tataranni et al., 2019; Li X and Zhou, 2010; Lim SC, et al., 2014; Yoon et al.,
et al., 2011). Treatment of proximal renal tubular cells with 2011. Statins: Adeli et al., 2017; Janic et al., 2019; Kuwahara et al.,

2008; Narumiya et al., 2004; Yoon et al., 2012. PPAR-γ agonists: Recombinant Klotho protein or peptides: Ali et al., 2020; Chen
Chen LJ, et al., 2014; Cheng et al., 2017; Huang KC, et al., 2014; TH, et al., 2013; Doi et al., 2011; Gupta et al., 2022; Hu et al., 2017;
Maquigussa et al., 2018; Shen et al., 2018; Zhang H, et al., 2008. Leon et al., 2017; Maltese et al., 2017; Mencke et al., 2017; Myung
mTOR inhibitors: Mizusaki et al., 2019; Tabibzadeh, 2021; et al., 2022; Oh et al., 2018; Prud’homme et al., 2020; Rao Z, et al.,
Tataranni et al., 2011; Zhao Y, et al., 2015. Vitamin D: 2019; Takenaka et al., 2018, 2019, 2020; Yang et al., 2015; Yuan
Haussler et al., 2020; Kuro-o, 2019; Lerch et al., 2018; et al., 2022; Zhong et al., 2020. Gene therapy: Arbel Rubinstein
Tsujikawa al., 2003. Antidiabetic drugs: Manrique et al., 2016; et al., 2021; Franco et al., 2021; Lin & Sun, 2015a, b; Ni et al., 2021;
Liu W, et al., 2017, 2021; Son et al., 2019; Wang Q, et al., 2019; Mencke et al., 2017; Shin et al., 2019; Xiang et al., 2021; Zeng et al.,
Yossef et al., 2020; Xue et al., 2019. Pentoxifylline: Navarro- 2019. Traditional medicines, nutraceuticals and supplements:
Gonzalez et al., 2018. Other: Zhou et al., 2022; Kang and Xu, 2016. Dehghani et al., 2019; Feng and Huang, 2022; Hsu et al., 2014;
Kamel et al., 2022; Long et al., 2018; Li SS, et al., 2018; Lim SW,
10.2 Drugs in Preclinical Development et al., 2019; Long et al., 2018; Ostojic and Engeset, 2021; Zhang P,
Numerous drugs, recombinant proteins (or peptides) and gene et al., 2016; Zhang XT, et al., 2020; Zhou et al., 2015. Exercise and
therapies have been tested in preclinical disease models for their sports: Amaro-Gahete et al., 2018, 2019a,b; Morishima and Ochi,
ability to boost Klotho levels (Table 3). Specific examples of the 2021; Tan et al., 2018.
administration of recombinant s-Klotho or peptides were
discussed before. Gene therapy has been performed with viral
vectors or plasmids, and cell therapy also appears feasible (Franco 10.4 The BBB and Treatment of
et al., 2021). In some cases, such as recombinant protein and gene Neurodegenerative Diseases
therapies, the feasibility, potential adverse effects, accessibility, The BBB (blood-brain barrier) blocks entry of the majority of
and high cost are likely to be limiting factors. drugs into the brain, and represents a challenge for the therapy of
Recombinant Klotho protein therapy has been successfully neurodegenerative conditions, as reviewed (Partridge, 2012;
applied to the treatment of renal, cardiovascular and Nilles et al., 2022). Some small lipid-soluble drugs cross by
neurodegenerative disease, as well as diabetes and cancer lipid-mediated diffusion.
(Table 3). Generally, these have been short-term study, with Other drugs may be shuttled by specific transporters. Large
injection of s-Klotho, the KL1 domain, or relevant peptides. Low drugs (usually proteins) generally do not cross, or transfer very
doses are usually effective. Recombinant protein has been delivered inefficiently, but transfer can be improved by engineering
systemically, or in the ventricles of the brain for neurological diseases. constructs for receptor-mediated transport. In addition,
Common food/diet components (e.g., astaxanthin, curcumin and neuropilin-1 appears to facilitate entry into the brain of some
resveratrol), and traditional medicines and/or their extracted cell-penetrating peptides, larger molecules, or viruses (Balistreri
compounds (e.g., baicalin, cordycepin, ginseng and ligustilide), et al., 2021; Prud’homme and Glinka, 2012; Ruoslahti, 2017; Zhao
have been examined and successfully induced Klotho (Table 3). L, et al., 2021).
Some of these are frequently incorporated into over-the-counter RAAS blocking drugs represent an interesting example,
nutraceuticals. In general, these compounds ameliorated or restored because they can be divided into BBB-crossing and non-
Klotho levels in rodent disease models where they are depressed. crossing types. For example, the AT1-receptor blockers
This work was performed in animal models, and it is largely losartan, irbesartan, olemesartan and eprosartan do not
unknown whether these substances can increase Klotho in humans. penetrate through the BBB, whereas valsartan, telmisartan and
candesartan do cross (Ho et al., 2017). Memory is improved in
10.3 Exercise and Fitness older adults treated with AT1 blockers, and users of the BBB-
Of major interest, physical exercise and sports are simple ways to crossing drugs performed better than those taking non-crossing
increase circulating Klotho, as reviewed by others (Amaro- drugs (Ho et al., 2017). The subjects receiving BBB-crossing drugs
Gahete et al., 2018). A single bout of exercise transiently also had significantly fewer white matter hyperintensities (WMH)
increases circulating Klotho (Tan et al., 2018; Morishima and upon imaging. Other investigators (Ohrui et al., 2004) reported
Ochi, 2021). The FIT-AGEING study examined the association similar findings in patients receiving BBB-crossing angiotensin-
between physical activity and fitness and plasma levels of converting enzyme (ACE) inhibitors. Those treated with the
s-Klotho in middle-aged sedentary adult people (Amaro- BBB-crossing drugs (captopril, perindopril) had a significantly
Gahete et al., 2019a,b). The authors reported that higher lower risk of developing Alzheimer’s disease, compared to those
activity and fitness associated with increased s-Klotho levels. taking non-crossing drugs (imidapril, enalapril). The protective
They assessed the effects of different courses of exercise over a mechanism is unclear. Can the BBB-crossing RAAS inhibitory
12-week period, and found that all types increased Klotho, as drugs increase Klotho in the brain or CSF? To our knowledge,
compared to no exercise. They report that higher s-Klotho there is no data in humans. In the cerebrum of spontaneously
associates with decreased fat mass and increased lean mass. hypertensive rats, however, valsartan treatment reduced cell
damage, as determined by ultrastructural changes such as
10.3.1 Summary of References for Drugs or apoptotic body formation (Li and Zhou, 2010). Valsartan
Treatments in Development treatment was associated with increased intra-cerebral
See also Table 3. GABA receptor agonist: Liu W, et al., 2021; expression of Klotho, as determined by RT-PCR,
Prud’homme et al., 2017a; Son et al., 2019; Wang Q, et al., 2019. immunohistochemistry, and Western blotting. This provides

some evidence, albeit limited, that valsartan can increase Klotho 11 CONCLUSION AND FUTURE
expression in the brain. Statins are thought to have at least some DIRECTIONS
neuroprotective effects. They can also be divided in BBB-crossing
and non-crossing. The lipophylic statins (e.g., atorvastatin, In this manuscript we explored the many facets of Klotho biology.
lovastatin, simvastatin) cross, and the hydrophilic ones do not The best characterized aspects relate to renal physiology, and
(Wood et al., 2010). In this case, simvastatin increased Klotho in especially phosphate and calcium homeostasis. The importance
the hippocampus of rats (Adeli et al., 2017). It is thus conceivable, of the FGF23/Klotho/FGFR interaction was evident in Klotho
but not yet certain, that both RAAS inhibitors and statins can deficient mice. The major outcomes were hyperphosphatemia
enhance Klotho expression in the human brain, and this might and hypervitaminosis D. The accelerated aging syndrome of these
protect against neurodegenerative diseases. mice was greatly ameliorated by a low phosphate diet. Rare
Some antidiabetic drugs (or at least members of these drug mutations of either Klotho or FGF23 in humans result in a
families) that enhance circulating Klotho levels in rodents can hyperphosphatemic syndrome, characterized by massive tissue
cross the BBB. This includes some GLP-1RAs (Pujadas and calcification and systemic inflammation. In chronic renal disease
Drucker, 2016), metformin (Moreira, 2014), and at least one of various etiology, as well as diabetes, Klotho production is
DPP-4 inhibitor (omariglipin) (Ayoub et al., 2018). DPP-4 decreased. It has been proposed that declining renal function, and
inhibitors increase GLP-1 (Deacon, 2019), the natural GLP- associated low Klotho, accelerate or aggravate many of the
1RA, which crosses the BBB. These antidiabetic drugs possibly pathologies found in old age. This may involve, for example,
ameliorate neurodegenerative disease in some patients (Mehan the precipitation of CPPs in tissue (Kuro-o, 2021).
et al., 2022), but a potential contribution of Klotho, to our It seems unlikely that all the manifestations of Klotho
knowledge, has not been examined. insufficiency are related to FGF23-induced signaling. Indeed, as
As shown in Table 3, several compounds derived from outlined in several sections, Klotho blocks major pathways that play
traditional medicines, or other sources, have been shown to a role in aging. This refers specifically to inhibition of TGF-β, Wnt,
increase Klotho expression in rodents. Interestingly, some of IGF-1 and NF-κB. These are complex pathways that impact on a vast
these compounds have been reported to cross the BBB, such as number of biological processes. Thus, regardless of other
astaxanthin, baicalin, cordycepin, ligustilide and resveratrol. mechanisms that may drive aging, the overactivation of these
Curcumin (Reddy et al., 2018) as well as a component of pathways is likely to be important. This is most evident in
ginseng, ginsenoside RG1 (Zhao YN, et al., 2018), also appear to fibrosis, inflammation and cancer, which are all countered by
cross the BBB at least to some extent. Ligustilide is of special interest, Klotho. Furthermore, Klotho activates antioxidant pathways, such
because it readily crossed the BBB and was protective in mice against as Nrf2 and FOXOs, which are thought to mediate antiaging effects.
ischemic brain injury and Alzheimer-like disease. In the ischemic In terms of future directions, there are several important avenues.
brain, NF-κB and inflammatory changes were reduced (Long et al., Klotho is described as an antiaging molecule, although there is no
2018). In the Alzheimer’s disease model, IGF-1/Akt/mTOR direct evidence that it delays aging in humans. This is one of the most
signaling was inhibited (Kuang et al., 2014). Ligustilide increased important questions to address, but it will require very long-term
Klotho in the choroid plexus. Downregulation of Klotho reduced studies. Klotho can be a marker of disease (kidney, brain or other), or
the protective effects. The neuroprotection appears to depend at a therapeutic molecule. In particular, the function(s) of Klotho in the
least in part on an enhancement of ADAM10 activity. As noted brain are not well defined, but neuroprotective effects are apparent.
previously, α-secretase cutting of APP by ADAM10 produces Klotho levels in the circulation or CSF could indicate susceptibility to
shedding of a large non-pathogenic segment, and prevents the disease. Multiple drugs appear to increase Klotho, but studies in
generation of small Aβ-forming peptides (Lichtenthaler et al., humans are scant. Does Klotho contribute to the therapeutic effects
2022). Ligustilide treatment enhanced ADAM10 activity resulting of some drugs used to treat hypertension, hyperlipidemia, diabetes or
in the higher production of both s-Klotho and non-amiloidogenic transplant rejection? Could some of these drugs ameliorate
APP. Aβ plaque formation was reduced in the ligustilide-treated neurodegenerative diseases by enhancing Klotho? This is all
mice. The authors (Kuang et al., 2014) concluded the enhancement unknown. Indeed, there have been practically no clinical
of s-Klotho and α-processing of APP both contributed to the investigations of Klotho-related treatment, outside of preclinical
amelioration of the neurodegenerative disease. work. Another important aspect is the role of Klotho as a tumor
In some cases, drugs that do not cross the BBB can ameliorate suppressor molecule. This is well documented in animals models,
Alzheimer’s disease, or other neurodegenerative conditions, by but needs to be developed in the clinical context. The slim clinical
indirect mechanisms. The improvement of hypertension, applications of Klotho research can perhaps be explained by the
hyperlipidemia, diabetes or renal function can all have a major limited understanding of its mechanisms of action, especially outside
impact on the brain. In young and aged mice, systemic delivery of the kidney. However, knowledge of the molecular and physiological
a Klotho-derived peptide that does not cross the BBB improved aspects of Klotho has progressed considerably in recent years, as
cognition and reduced neurodegenerative pathology (Leon et al., outlined in this review.
2017). Others showed that systemic delivery of either s-Klotho or For therapy, several approaches are feasible, as described in this
only the KL1 domain also improved cognition (Gupta et al., manuscript. Clinical research focused on any of these approaches
2022). It is unclear how these beneficial Klotho-based effects are would be an important contribution to the field. The most direct
mediated, but penetrance into the brain does not appear to be approach is the administration of recombinant Klotho or peptides.
required, and this merits further investigation. In view of the relatively short half-life of these products, there is a

need to produce long-acting constructs, or slow-release supervised some of the studies reported and edited the
formulations. There is little evidence from preclinical work that manuscript. All authors reviewed and approved the content of
Klotho administration is toxic, at least when restoring normal this manuscript.
levels. However, the long-term effects of supra-normal levels are
not known. Gene therapy is also feasible, but much more
challenging. In addition to some clinical prescription drugs, FUNDING
there are also commonly available over-the-counter medicines,
supplements, or nutraceuticals that increase Klotho. This is based The work performed by the authors in this manuscript was
on animal work, and it would be of much interest to examine the funded by the Juvenile Diabetes Research Foundation
possibility of Klotho enhancement in humans. In the case of International (grant numbers 2-SRA-2015-64-Q-R and 2-SRA-
neurodegenerative diseases it is relevant that some drugs, 2018-497-A-B), the St. Michael’s Hospital Foundation (Toronto,
including some traditional medicines, cross the BBB and might Canada), the Keenan Research Centre for Biomedical Science
increase Klotho in the brain. Finally, exercise and the long-term (Toronto, Canada), the Canadian Diabetes Association (grant
maintenance of fitness are simple methods to increase Klotho. In number OG-3-13-4066), and the National Science Foundation of
conclusion, most of the studies on Klotho have been performed in China (grant numbers 81570518, 81630020, 81800751).
animal disease models, and a considerable amount of work has to
be performed to introduce Klotho therapy into the clinic. This
review provides a framework of mechanisms and findings that ACKNOWLEDGMENTS
favor the clinical investigation of Klotho-based treatments.
The authors thank Dr. Yelena Glinka, Dr. Anna Toulina, Dr.
Wenjuan Liu and Dr. Dong Ok Son for their expert work and
AUTHOR CONTRIBUTIONS advice in performing experiments from our laboratories. Some
images in the figures were from Servier Medical Art image (smart.
GP wrote the manuscript, MK performed Klotho-related servier.com), obtained under the Creative Commons Attribution
experiments and prepared figures for this manuscript, QW 3.0 Unported License.
[3H]-Muscimol within Rat Kidney. Pharmacology 36 (6), 390–395.

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Dihydroartemisinin suppresses renal fibrosis in mice by inhibiting DNA- any commercial or financial relationships that could be construed as a potential
methyltransferase 1 and increasing Klotho. Acta Pharmacol. Sin. doi:10. conflict of interest.
1038/s41401-022-00898-3
Zhou, X., Yang, Q., Xie, Y., Sun, J., Hu, J., Qiu, P., et al. (2015). Tetrahydroxystilbene Publisher’s Note: All claims expressed in this article are solely those of the authors
glucoside extends mouse life span via upregulating neural klotho and and do not necessarily represent those of their affiliated organizations, or those of
downregulating neural insulin or insulin-like growth factor 1. Neurobiol. Aging the publisher, the editors and the reviewers. Any product that may be evaluated in
36 (3), 1462–1470. doi:10.1016/j.neurobiolaging.2014.11.002 this article, or claim that may be made by its manufacturer, is not guaranteed or
Zhu, H., Gao, Y., Zhu, S., Cui, Q., and Du, J. (2017). Klotho Improves Cardiac endorsed by the publisher.
Function by Suppressing Reactive Oxygen Species (ROS) Mediated
Apoptosis by Modulating Mapks/Nrf2 Signaling in Doxorubicin-Induced Copyright © 2022 Prud’homme, Kurt and Wang. This is an open-access article
Cardiotoxicity. Med. Sci. Monit. 23, 5283–5293. doi:10.12659/msm.907449 distributed under the terms of the Creative Commons Attribution License (CC BY).
Zou, D., Wu, W., He, Y., Ma, S., and Gao, J. (2018). The role of klotho in The use, distribution or reproduction in other forums is permitted, provided the
chronic kidney disease. BMC Nephrol. 19 (1), 285. doi:10.1186/s12882-018- original author(s) and the copyright owner(s) are credited and that the original
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Zubkiewicz-Kucharska, A., Wikiera, B., and Noczyńska, A. (2021). Soluble No use, distribution or reproduction is permitted which does not comply with
Klotho Is Decreased in Children With Type 1 Diabetes and Correlated these terms.

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published: 12 July 2022

Pathobiology of the Klotho Antiaging

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1 INTRODUCTION 1) FGF23-dependent phosphate, calcium and vitamin D

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4 KLOTHO INTERACTIONS WITH FGF23

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6 KLOTHO FUNCTIONS INDEPENDENT OF

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TABLE 1 | Klotho insufﬁciency and related pathologies.

Organ/system Disease or lesion References

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TABLE 2 | Examples of current clinical drugs that increase Klotho.

Category (name) Usual Clinical applications References

Renin-angiotensin-aldosterone inhibitors (losartan, valsartan) Hypertension Janic et al. (2019)

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Category (name) Target diseases References

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[3H]-Muscimol within Rat Kidney. Pharmacology 36 (6), 390–395.

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