Antiaging Properties of the Klotho Protein

Aging is an inevitable fact of life. It affects all organs and tissues, leading to impaired function, major debilitating diseases and death. Aging can be slowed by lifestyle changes such as exercise and diet. It is accelerated by many well-known negative factors. This includes smoking, obesity, hyperlipidemia, hypertension, chronic infectious diseases, anti-cancer drugs, radiation and exposure to environmental toxins. In diabetic subjects, high or poorly controlled glucose levels markedly accelerate aging. Similarly, renal disease and associated hyperphosphatemia (phosphatopathy) injure cells and accelerate aging [1].

α-Klotho (denoted here Klotho or KL) is an antiaging agent that is expressed in the kidneys, brain, endocrine organs (e.g., pancreatic β cells, parathyroid glands), blood vessels, skin, and other tissues [2]. It is also expressed by peripheral blood cells. Klotho is an obligate coreceptor for fibroblast growth factor 23 (FGF23), an endocrine FGF (eFGF) produced in bone, but it has several other functions, as reviewed by [3]. Klotho-deficient hypomorphic (KL^kl/kl) or null (KL^-/-) mice exhibit rapid aging. This syndrome includes stunted growth, hyperphosphatemia, hypercalcemia, cardiac disease, vascular disease (especially calcification), osteopenia, emphysema, sarcopenia, cognitive deficits, multi-organ atrophy and a short lifespan [4,5]. In contrast, overexpression of Klotho in transgenic mice [6], or induced by gene transfer [7], mitigates several features of aging, and increases lifespan by as much as 20 to 30%. There is a membrane-bound form; as well as a soluble form (s-Klotho) found in the blood and the CSF. In humans, s-Klotho declines with age and in several chronic diseases. Furthermore, low Klotho levels are associated with an increase in mortality. In this review, we focus primarily on the mechanisms by which Klotho delays aging.

There are many theories of aging, but the underlying molecular and cellular mechanisms are still not well defined. Different species age at different rates, suggesting the existence of a poorly understood biological clock [8]. We do not see 80-year old cats or dogs, but many humans surpass that age. The Greenland shark lives hundreds of years. Diverging lifespans (the time from birth to death) most likely represent species evolution in response to the environment, predators, and the availability of food. In order for a species to survive, it has to reproduce at a rate sufficient to match or exceed the death rate. Animals such as mice and rabbits have a short lifespan, but reproduce at a very fast rate. Humans live much longer and reproduce relatively slowly.

Humans are exceedingly complex multi-cellular organisms. Scientists are very far from elucidating all the molecular, cellular and whole organ aspects. To complicate matters, aging affects practically all cells and organs. Different organs can age at a different rate and in different ways. Some connective tissue changes appear irreversible. In view of the biologic complexity, it is not surprising there is no generally accepted theory of aging. Instead, to address this issue, researchers have described a number of hallmarks of aging [9]. There are currently twelve hallmarks, but this could increase. These hallmarks are mostly based on reductionist studies performed in various species (yeast, nematodes, insects and mice), and to a much lesser extent in humans.

As mentioned previously, Klotho action mitigates several hallmarks of aging (Figure 1), and ameliorates numerous age-related pathologies (Figure 2). It is beyond the scope of this manuscript to cover all these aspects in detail. However, concerning the pathobiology of Klotho, there have been a number of relevant reviews published by others and us [3,5,10,11,12,13,14,15,16,17]. Here, we list the Klotho activities that appear most relevant to aging, whether or not they are linked to the 12 hallmarks. These findings were mostly obtained from preclinical work, but they are in accord with available clinical findings. This is a brief overview of these findings, and several of these features are discussed in subsequent sections of the manuscript.

The molecular aspects of Klotho and its function as a coreceptor for FGF23 have been extensively studied, as reported by several researchers [5,10,52,53,54,55,56,57,58,59,60,61,62,63]. Klotho is expressed on the membrane as a single-pass protein, with a very short cytoplasmic segment (10 aa). In humans it has 1012 aa (130 kD). It has two extracellular domains of similar size—denoted KL1 and KL2. A soluble form of Klotho (s-Klotho) is produced by shedding. This soluble form is also termed processed Klotho (p-KL) by some authors [59]. It is generated by the cleaving action of extracellular proteases, principally ADAM10 and ADAM17 (α-secretases) [58,60]. The released KL1/KL2 segment appears to be the main (and perhaps only) circulating form of Klotho. This is consistent, for example, with the depletion of circulating Klotho observed in mice treated with secretase inhibitors.

However, a smaller form generated by alternative splicing has been described. It encodes KL1 and an additional C-terminal tail of 14 amino acids (but not KL2) [59]. It is termed secreted Klotho (sometimes confusingly abbreviated s-KL). Its existence has been questioned by some authors [61]. These researchers reported that the alternative Klotho mRNA (putative secreted Klotho) has premature termination codons that mark it for degradation by nonsense-mediated RNA decay (NMD). They could not identify production of the protein in vitro. To our knowledge there has been no other publication about Klotho NMD. Interestingly, unlike mice and humans, rats are devoid of this alternative splice variant. In any event, the secreted form of Klotho can be produced by gene therapy vectors, which presumably carry only the coding sequence (no RNA splicing) [7,59]. This allows selective expression of the secreted KL1 domain in vivo, and this has been linked to increased longevity and health in mice [7].

Crystal structure analysis demonstrated that Klotho binds to FGF receptors to produce a high affinity receptor for FGF23 [53]. In the absence of Klotho, FGF23 has low affinity for FGFRs. Klotho has affinity for FGFRs 1c, 3c and 4 [53,62], although FGFR1c appears to be employed the most often. Klotho attaches to FGFR1c by an extension of its KL2 domain. FGF23 binds into a groove formed by components of KL1, KL2 and the FGFR [53,63]. Thus, KL2 is required for receptor assembly and FGF23 binding. The KL1 domain also contributes to FGF23 binding, but does not interact directly to FGFRs.

Importantly, KL1 (with or without KL2) performs many Klotho functions that appear FGF23-independent. This includes blockade of TGF-β, Wnt ligands, insulin-like growth factor 1 (IGF-1) and other mediators. For example, a KL1 domain construct exerted anti-cancer activity, but did not promote FGF23 signaling [64]. Related to this issue, Roig-Soriano et al. [59] compared in vivo gene transfer-based treatment with processed Klotho (KL1/KL2 segment) versus secreted Klotho (KL1 domain only). The processed Klotho differed markedly in that it increased FGF23 production in bone, and altered bone microstructure. It also perturbed phosphate and calcium homeostasis. The secreted Klotho did not have these effects. This is consistent with soluble KL1/KL2 acting as a FGF23 coreceptor, although this question was not examined.

In accord with in vitro and in vivo studies, s-Klotho (soluble KL1/KL2) can attach to FGFRs, and function as a coreceptor for FGF23 [53,63,65,66]. This is supported by the finding that s-Klotho protein injection into Klotho hypomorphic mice increased urinary phosphate excretion [67]. In wild-type mice, s-Klotho similarly increased phosphate excretion and lowered serum phosphate [63]. In vitro, s-Klotho enhanced FGF23-induced signaling in Klotho-negative HEK293 cells [53]. Co-stimulation with soluble Klotho appears weaker than with membrane Klotho. Of note, free FGF23 can also bind directly to free s-Klotho (KL1/KL2) at relatively high affinity [56,57,68]. Presumably, the FGF23/s-Klotho soluble complex can then attach to membrane-bound FGFRs and induce signaling. However, the extent to which s-Klotho promotes FGF23 signaling under ordinary physiological conditions remains unclear.

Klotho physiology in the kidney is the area that has been the best studied and characterized [5,54,55,69,70,71]. Upon binding of both Klotho and FGF23, the activated FGFR signals through multiple pathways, notably PI3K/Akt, Ras/MAPK/ERK and phospholipase Cγ (PLCγ). The activated FGFR1c receptor contributes to phosphate and calcium exchange. In the proximal renal tubule, inorganic phosphate (Pi) re-absorption is reduced (phosphaturic effect), through inhibition of the sodium-phosphate transporters NPT-2a and NPT-2c [53]. Klotho’s action in the distal tubule is different. There, Ca²⁺ resorption is enhanced by TRPV5 channels. Of importance, FGF23/Klotho/FGFR1c regulates vitamin D by inhibiting 1α-hydroxylase in the proximal renal tubule. This reduces the synthesis of active vitamin D (1,25(OH)₂D₃)—termed calcitriol. There is also increased enzymatic inactivation of calcitriol, but the regulation of this is not totally resolved [71].

Lack of either Klotho or FGF23 in mice results in hyperphosphatemia, hypercalcemia, 1α-hydroxylase overactivity and hypervitaminosis D. A low phosphate diet improves disease, despite the fact that vitamin D increases further [5]. In humans, Klotho deficiency is most commonly the result of acute or chronic kidney failure [55]. Note that contrary to Klotho hypomorphic mice, these persons usually have low vitamin D levels. This is consistent with advanced renal disease.

Rare human mutations (usually autosomal recessive) cause a severe loss of either FGF23 or Klotho [5,72,73]. Nearly all cases involve FGF23. Very infrequently, low FGF23 levels are caused by anti-FGF23 antibodies, in an autoimmune process. To our knowledge, a Klotho loss of function mutation has only been reported in one person [74]. In all these different cases, the subjects have hyperphosphatemia. Vitamin D levels may also be high. At a young age, these patients develop massive calcification in soft tissues (tumoral calcinosis); as well as calcification of the arteries, and calcification in multiple organs and tissues. They have bone and dental anomalies, and systemic inflammation. To our knowledge, there has not a been a detailed analysis of aging over a long period of time in these individuals. However, hyperphosphatemia and systemic inflammation are factors that can accelerate aging. Note that subjects with FGF23 deficiency can still produce Klotho, which has FGF23-independent functions. In usual end-stage renal disease Klotho levels are low, and these persons have hyperphosphatemia and multiple other anomalies [5,55].

Some clinical FGF23 overproduction syndromes have been reported. These are due to mutations that increase FGF23 levels, or to FGF23-secreting tumors [73,75]. As expected, these individuals develop hypophosphatemia. Similarly, a translocation causing excessive Klotho production (single case reported) was associated with hypophosphatemia [76].

In addition to these findings, several Klotho genetic variants have been described [77,78,79,80,81,82]. The best characterized is denoted KL-VS haplotype. It consists of six single nucleotide polymorphisms (SNP) in strict linkage disequilibrium [77]. The KL-VS haplotype carries two mutations in the coding sequence that produce amino acid substitutions (F352V and C370S). Its occurrence varies in different populations, and it is present in 15% of Caucasians. In neuropathologies and other conditions it is generally protective in the heterozygous state, rather than in the rarer homozygous state where it can be detrimental. KL-VS heterozygosity associates with increased lifespan [78,80]. It has been linked to improved cognitive functions, as well as reduced amyloid-β (Aβ) and risk of Alzheimer’s disease [77,79,80]. Notably, KL-VS heterozygosity is associated with reduced neuroinflammation and neurodegeneration, at least in some of the subgroups that were examined [79]. KL-VS heterozygosity also appears to be protective against cardiovascular disease [81]. Several other Klotho polymorphisms have been associated with disease, but have not been studied as thoroughly as KL-VS. For example, some Klotho genetic variants altered disease expression in either a positive or negative way as related to cognition [77] and diabetes [82]. A caveat is that the beneficial effects of KL-VS heterozygosity have not been confirmed in all studies. The role of the other variants requires more extensive investigation. In all cases, the protective mode of action is not well elucidated.

Klotho blocks TGF-β action, as we have previously reviewed [16]. This is particularly important because overexpression of TGF-β contributes to aging in several ways. This cytokine, and other members of the TGF-β family, can promote cellular senescence, cell death, fibrosis, immune dysfunction, loss of muscle mass, as well as cancer progression [106,107,108,109]. Indeed, TGF-β is involved in a vast number of cellular responses. The TGF-β family includes 33 members. TGF-β is the best characterized, and it signals by a canonical pathway and numerous noncanonical pathways [110,111,112]. It is secreted in latent form, binds to connective tissue components, and is activated primarily by interaction with integrins, although other mechanisms apply. The three isoforms of TGF-β (TGF-β1 is the most abundant) all bind to the same membrane-bound signaling receptor. It consists of two chains; the type 1 chain is denoted TβRI (ALK5) and the type 2 chain TβRII. In the initial interaction, TGF-β binds to TβRII and TβRI to create a serine/threonine kinase complex. TβRII phosphorylates TβRI, which in the canonical pathway phosphorylates Smad2 and Smad3. These two Smads form a complex with Smad4 (the common Smad) that translocates into the nucleus. It then binds to DNA and regulates the transcription of numerous genes.

The TGF-β signaling receptor also activates key noncanonical pathways including ERK, JNK, p38 MAPK, PI3K/Akt, Rho-like GTPases and NF-κB [110,112]. Moreover, it can crosstalk with other pathways influencing a large proportion of cell functions, including other TGF-β family cytokines (BMPs, Nodal, myosin), Wnt, Notch, Hedgehog, Hippo (TAZ/YAP) and JAK/STAT. It can also crosstalk with vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and hepatocyte growth factor (HGF). This demonstrates the exceedingly high number of pathways that can be modified when increasing or decreasing TGF-β activity.

TGF-β is a key regulator of the immune system. It is secreted by regulatory T cells (Treg), macrophages and several other cell types. It exerts immunosuppressive or regulatory functions on dendritic cells, macrophages, B lymphocytes, effector T lymphocytes, NK cells and neutrophils [107,113]. As related to cancer, TGF-β acts as a tumor suppressor in the early stages of neoplasia, but at later stages it promotes cancer progression and metastasis [109].

Klotho inhibits TGF-β by binding to the signaling TGF-β receptor [39,41]. Indeed, it has been reported to attach to the TβRI and TβRII chains of the receptor. These inhibitory effects have been demonstrated in vivo, especially against fibrosis. For instance, Klotho or a short Klotho peptide (KL1 domain) prevented renal fibrosis in mice [114]. The inhibition of TGF-β family members by Klotho might be applicable, for example, to pulmonary fibrosis, chronic renal disease, diabetes, cirrhosis of the liver, systemic sclerosis and sarcopenia. However, applications are still at a preclinical stage.

Sarcopenia (loss of muscle mass and/or function) is a feature of old age, inflammation/inflammaging, several chronic diseases and cancer. Klotho deficient mice (kl/kl or knockout) have sarcopenia and, in humans, low Klotho levels associate with sarcopenia. This subject has been recently reviewed by others [42,115], and is only briefly covered in this manuscript. The pathogenesis of sarcopenia is complex and not fully understood. The underlying mechanisms likely differ considerably depending on the clinical context, such as aging, obesity, kidney disease, cancer and other conditions. Obesity (sarcopenic obesity) [115] and chronic kidney disease [116] are common factors that induce sarcopenia. Both are associated with decreased s-Klotho levels. Importantly, Klotho overexpression in mice (by viral gene transfer) ameliorated muscle mass and structure, although this appeared to be age dependent [45]. Interestingly, this treatment had an impact on the expression of all the groups of genes related to the hallmarks of aging [45].

The action of TGF-β and myostatin (another TGF-β family member) appears important, at least in some experimental models of sarcopenia. Myostatin reduces muscle mass, as occurs in senile muscle atrophy and cachexia [117]. Ohsawa et al. [41] reported that Klotho inhibits multiple members of the TGF-β family that can reduce muscle mass. This involved TGF-β1, myostatin, GDF11 and activins. This work was performed in vitro; however, the injection of a TGF-β inhibitory drug ameliorated muscle mass in Klotho knockout mice and old wild-type mice.

Other authors have reported that Klotho ameliorates muscle regeneration and healing in response to injury [118]. In renal failure, excess FGF23 and low Klotho induce muscle wasting [119]. It has been proposed that this is due to inflammation; particularly NF-κB activation and the release of inflammatory cytokines. Furthermore, high levels of FGF23 suppress the levels of Klotho and likely negate its activity.

The findings outlined above suggest that Klotho has potential for the treatment of sarcopenia. However, it is still unknown whether it can be applied to human sarcopenia treatment.

Klotho has major antioxidant properties, mainly by activating Nrf2 and FoxO. This promotes the expression of several antioxidant enzymes, including superoxide dismutase (SOD), glutathione (GSH)-associated enzymes, catalase, and the thioredoxin system [35,102,120]. Klotho activated Nrf2 in diseases of the kidneys, the brain and the cardiovascular system [37,121,122,123,124].

In the case of FOXO proteins, Klotho inhibits the IGF-1 receptor, which abrogates IGF-1 inhibition of FOXO. Then, the FOXO proteins migrate into the nucleus and mitigate oxidative stress through enzymes including catalase and manganese SOD [35]. For instance, Klotho protected rat hearts from ischemia–reperfusion injury (IRI) [38]. This depended on the inhibition of the IGF1R/PI3K/AKT pathway.

As mentioned previously, Klotho has been found to protect cells against senescence. This has been observed in kidney tubules [24], cardiovascular system [23,123], lungs [124], brain [25,125]; pancreatic β cells [26], and other sites.

Classically, cellular senescence was recognized as a feature of telomere attrition, especially in cells dividing in culture. However, cell senescence is also a response to cell injury, which can occur in many situations [126]. For example, it can be triggered by oxidative stress, mitochondrial dysfunction, DNA damage, ER stress, oncogene activation, cancer chemotherapy, inflammatory cytokines, and TGF-β. The two classical features are cell cycle arrest (G1/S or G2 phase), and the senescence-associated secretory phenotype (SASP) [127]. Senescence-associated β-galactosidase (SA-β-gal) is a marker that is frequently used to identify senescent cells. Activation of some pathways (especially p16 and p53-p21) leads to permanent cell-cycle arrest, although these cells can maintain some functions and have SASP. The SASP consists of the secretion of chemokines, inflammatory cytokines, growth factors and other mediators. However, the senescence markers and composition of the SASP can vary considerably from one cell type to another.

Cells that do not normally divide can nevertheless show other features of senescence, although this can be more difficult to establish. We examined senescence in pancreatic β cells [26]. Of interest, we observed that old mice had increased numbers of senescent β cells. Furthermore, the β cells of these old mice showed severe downregulation of Klotho expression. We examined β-cell senescence in vitro with the INS-1 pancreatic β cell line [26]. Senescence was induced by incubation with doxorubicin. The senescent cells displayed marked loss of Klotho expression. However, the addition Klotho to the cultures protected these cells against senescence. It improved mitochondrial function and reduced ROS production. Klotho also improved insulin secretion. These findings suggest that Klotho has a protective role in the endocrine pancreas, in accord with our previous studies [90,128].

Pathology involving the arteries is a major negative aspect of aging. It is a frequent cause of death. Arterial calcification and atherosclerosis occur gradually with age. The occurrence of early (premature) vascular aging is precipitated by either smoking, diabetes, hypertension or chronic kidney disease. In cases of advanced renal failure, Klotho insufficiency leads to hyperphosphatemia and markedly increased FGF23 levels. These extremely high levels of FGF23 can activate FGFR4 in the absence of Klotho [129]. High FGF23 levels have multiple adverse effects such as endothelial-cell dysfunction, ventricular hypertrophy, heart failure and increased mortality [129]. In contrast to FGF23, high Klotho levels associate with reduced heart failure [48].

In Klotho-deficient mice, calcification of the arteries occurs rapidly and is a prominent feature [4]. In humans over 70 years old, calcium deposition in multiple vessels is common; notably in the aorta, peripheral arteries, coronary arteries and cerebral arteries [130]. In some cases, calcium accumulates mainly in the media of arteries (Monckeberg’s medial calcific sclerosis) [131]. However, calcium is often deposited in atherosclerotic plaques, including the most clinically relevant sites such as the coronary and cerebral arteries.

Atherosclerosis associates with hyperlipidemia, but inflammation is an important contributing factor [132]. Cholesterol crystals accumulate in the plaques, and these crystals are phagocytosed by macrophages. This results in the activation of the NLRP3 inflammasome. In this situation, thioredoxin-interacting protein (TXNIP), ROS and calcium deposits can probably all activate the inflammasome. Subsequent to this, plaque erosion or rupture leads to thrombosis and vessel occlusion [133].

Recently, in a clinical study, Hellou et al. [134] reported that circulating Klotho levels inversely correlated with calcification of the aorta and iliac arteries. Furthermore, low Klotho was independently associated with increased mortality. Interestingly, Klotho was identified in the wall of blood vessels, and circulating Klotho might not be the only factor. In several previous studies, others also reported that Klotho insufficiency associates with calcific vasculopathy and/or atherosclerosis [135,136,137,138]. Klotho appears to protect against vascular disease by reducing calcification, inflammation and endothelial cell injury.

Diabetes promotes atherosclerotic disease in arteries of various sizes, but arterioles and capillaries can also be involved. Indeed, diabetic retinopathy (DR) is characterized by microangiopathy of the retina. In advanced DR cases (proliferative DR) there is angiogenesis, hemorrhage and fibrosis. This can progress to blindness. In the retina, Klotho is produced locally and appears essential to maintain normal function [139]. The findings suggest that Klotho mitigates DR by preventing epithelial–mesenchymal transition (EMT), ROS production, vascular endothelial growth factor (VEGF) release, and cell death by apoptosis [140,141]. To our knowledge, its use for clinical treatment has not been reported.

Klotho is also relevant to age-related macular degeneration (AMD). TGF-β2 (produced in the eye) is thought to be a major factor in the pathogenesis of AMD [142]. Increased vitreous TGF-β2 likely induces retinal fibrosis. In mice, intravitreal injection of Klotho protected against TGF-β2-induced EMT, and retinal epithelial-cell degeneration [142]. Klotho treatment ameliorated cell senescence and EMT. There was reduced cytosolic and mitochondrial oxidative stress. These reports in DR and AMD suggest that Klotho could useful in the treatment of some retinopathies.

Klotho is expressed in the skin and other epithelial tissues [2]. Strong positive staining was seen in the epidermis and skin appendages. The skin is a major target of aging, and subject to injury by ultraviolet (UV) radiation and many other factors. Cells may undergo senescence and apoptosis. It is a major target of inflammaging, as well as a wide variety of inflammatory diseases [143]. Despite this, there have been very few studies of Klotho function in the skin. Klotho protected human keratinocytes against UV radiation, possibly by inhibiting NF-κB [144]. It prevented endothelial-to-mesenchymal transition (EndMT) [145], and this also appeared to be related to diminished NF-κB activation. Recently, Humble et al. [146] examined the effect of a Klotho-containing serum on photoaging in a pilot study. The participants experienced improvement in photoaging, wrinkles and other changes. Larger studies are required to confirm these observations.

Age is a major factor in the occurrence of cancer. Some hallmarks of aging are relevant to cancer—especially genomic instability (DNA damage), epigenetic alterations and chronic inflammation. However, cancer cells resist telomere attrition and cell cycle arrest (senescence).

Several studies have confirmed that Klotho exerts potent anti-tumor activity. This has been extensively reviewed by others [15,43,147,148,149], and is only briefly addressed here. Indeed, Klotho inhibits several pathways that promote cancer progression, including Wnt, IGF-1, TGF-β, NF-κB and others. Klotho expression is frequently suppressed in malignant tumors, and tumors expressing higher levels have a more favorable prognosis. Reduced Klotho expression is often related to increased promoter methylation. Klotho is protective in several types of cancer, such as breast, pancreas, colon and liver. Importantly, inducing the expression of Klotho, or treatment with s-Klotho, suppressed the growth of cancer cells, as reviewed [149]. Klotho suppressed cancer cell proliferation, invasion and colony formation. For instance, Abramovitz et al. [64] found that Klotho expression was low in pancreatic carcinoma. Klotho treatment, or overexpression of Klotho, reduced pancreatic cancer cell proliferation, both in vitro and in vivo. It countered IGF-1 and FGF pathways. Injection of the Klotho KL1 domain mimicked the anti-cancer activity of the larger s-Klotho (KL1/KL2). Overall, these studies highlight the potential of Klotho as an anti-cancer agent.

The role of Klotho in the CNS is not precisely elucidated, but there is extensive evidence of neuroprotection. With age, Klotho levels decline in the CSF, and its expression is reduced in the brain. Generally, low levels of Klotho associate with decreased cognitive abilities. The protective function of the KL-VS haplotype in heterozygous form was discussed previously (Section 6). More direct evidence of Klotho protection has been observed in human cerebral organoids. Forced expression of Klotho by these cells decreased neuronal senescence [150]. Importantly, Klotho protected cultured cortical neurons against Aβ toxicity; reducing neuronal apoptosis and degeneration [151]. Interestingly, these investigators observed that Klotho is expressed mostly in the axons of cortical neurons [151]. This work establishes that Klotho has direct neuroprotective effects. In cultures of neural hippocampal precursor cells, others observed that Klotho augmented neuronal differentiation and decreased apoptosis [152]. From an in vivo point of view, Klotho protein injections in nonhuman primates improved their performance in cognitive tests [153].

Indeed, Klotho-dependent neuroprotection has been reported in many preclinical studies [154,155,156,157,158,159,160,161]. It exerts anti-inflammatory and neuroprotective effects [157]. It also ameliorates myelination in the CNS, which is particularly relevant to multiple sclerosis [154]. Recently, Roig-Soriano et al. [7] delivered secreted Klotho in mice with an adeno-associated viral (AAV9) vector. It was administered by combined i.v. and intra-cerebroventricular (ICV) injection. This resulted in increased systemic and cerebral Klotho production. The injected mice showed several benefits, including increased longevity, reduced muscle fibrosis, ameliorated muscle regeneration response and, in the brain, increased cellular markers of neurogenesis.

In Alzheimer’s disease, neuroinflammation and related inflammasome activation are likely key pathogenic factors [162,163,164,165,166,167]. Notably, Aβ and hyperphosphorylated Tau protein, which are major molecular targets, promote the activation the NLRP3 inflammasome [167,168]. Moreover, other molecular targets and other inflammasomes are most likely involved. Klotho reduces neuroinflammation. Notably, it suppresses ROS, TXNIP, NF-κB activation, NLRP3 inflammasome action, and neuronal cell death.

The ability of Klotho to suppress aging has been known for almost thirty years. These antiaging properties are still not fully delineated, but cannot be ascribed to a single mechanism. Instead, we note several features that match the current hallmarks of aging, as reported in the literature. In mice, overexpression of Klotho modified the expression of all the groups of genes related to the hallmarks of aging. Furthermore, Klotho mitigates several major pathologies that are likely multifactorial and not easily explained by a single hallmark. Indeed, the antiaging properties of Klotho are exceptional. This is completely consistent with the fact that Klotho inhibits several pathways that have long been known to promote aging, such as TGF-β, IGF-1, NF-κB and Wnt.

In addition to this, the regulation of phosphate/calcium homeostasis is important. Klotho deficiency causes hyperphosphatemia, which is not one of the hallmarks but can mediate cellular injury. Other aspects such as protection against DNA damage, telomere attrition and stem cell depletion agree well with the hallmarks. However, in the case of Klotho, some of these aspects have not been examined extensively, and the actual mechanisms involved require more investigation. A major action of Klotho is the reduction in cellular senescence, which has been identified repeatedly. Another recurrent finding is the amelioration of mitochondrial function, and depletion of ROS. These are all important components of the aging hallmark classification.

Protection against inflammation might be the most important feature. Chronic inflammation is one of the major hallmarks of aging. Furthermore, inflammation exacerbates the other hallmarks. Examining the literature, it is evident that most of the diseases that are ameliorated by Klotho have a strong inflammatory component. In fact, Klotho can counteract both acute and chronic inflammation. This is consistent with Klotho’s ability to block NF-κB and the NLRP3 inflammasome. In addition, inflammation contributes to cell death by several pathways. In this regard, Klotho might also be effective against sepsis, and associated septic shock that has a high rate of fatal outcome.

Another key point of Klotho is its anti-fibrotic action. It has been shown to prevent fibrosis in major organs (kidneys, heart, lungs and liver), mainly through the inhibition of TGF-β and Wnt. At advanced stages fibrosis can lead to organ failure, and this is much more likely to occur in aged individuals. The blockade of TGF-β and myostatin also appear to protect against sarcopenia, at least in experimental models. Sarcopenia is a very common morbidity of aging, obesity, chronic diseases and cancer. The inhibition of TGF-β may also be relevant to the treatment of some retinopathies (e.g., diabetic, AMD). Finally, the antitumor functions are well documented, involve several mechanisms, and appear likely to be clinically applicable.

We examined several factors that increase or decrease Klotho. For example, hyperglycemia is a negative factor, and most anti-diabetic drugs increase Klotho. NF-κB (as in inflammation) and TGF-β (as in fibrosis) are other negative regulators of Klotho expression of clinical importance. They are potentially amenable to Klotho therapy. Practically all current drugs or other treatments that increase Klotho also have many other effects. In these cases, it can be extremely difficult to determine the contribution of Klotho. A major limitation remains the lack of clinical investigation of direct Klotho therapy, and this should be a prime goal of future research.