Aging is associated with changes in almost all tissues and organs of the body, resulting eventually in debilitating and/or fatal conditions such as cardiovascular disease, chronic organ failure, neurodegeneration and cancer. Aging occurs at varying speed in different species, suggesting some poorly defined biological clock. The molecular mechanisms underlying aging are intensely studied, but still not well understood. However, the pathogenesis of age-associated diseases is likely multi-factorial (Franceschi et al., 2018). In humans, a limited number of genes have been clearly linked to accelerated aging as seen in progeria (a rare condition) (Coppede 2021) or, conversely, associated with longevity (Javidnia et al., 2022). The subject of this review is Klotho (kl), which is an antiaging gene, and the corresponding protein α-Klotho (henceforth denoted Klotho or KL). The gene was first identified in mice in 1997 (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 (Kl ^kl/kl ) (Kuro-o, et al., 1997) or full knockout of the Klotho gene (Kl ^−/− ) (Tsujikawa et al., 2003). Klotho-deficient mice exhibit stunted growth, renal disease, hyperphosphatemia, hypercalcemia, vascular calcification, cardiac hypertrophy, hypertension, organ fibrosis, multi-organ atrophy, osteopenia, pulmonary disease, cognitive impairment and short lifespan (Bian et al., 2015; Erben and Andrukhova, 2017; Kinoshita and Kawai, 2016; Kuro-o et al., 1997, 2010, 2019, 2021). Overexpression of the gene has the opposite effects, lengthening survival (Kurosu et al., 2005).

Klotho insufficiency appears to play a role in human aging and, specifically, in many of the diseases that are associated with aging. Klotho expression declines with age, renal failure, diabetes and neurodegenerative disease. The age-related decline in serum levels appears to be similar in men and women; and reference values have recently been reported (Espuch-Oliver et al., 2022). Notably, a recent study of American adults showed that low serum Klotho levels correlate with an increased all-cause death rate (Kresovich and Bulka, 2021).

Klotho can exist as a membrane-bound coreceptor for fibroblast growth factor 23 (FGF23) (Urakawa et al., 2006), or a soluble endocrine mediator with many functions (Figures 1, 2) (Dalton et al., 2017; Kuro-o, 2017). Age-related deterioration of renal 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 elimination (phosphaturic effect). Remarkably, independent of FGF23, it inhibits at least four pathways that have been linked to aging in various ways. Klotho blocks or inhibits transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), nuclear factor κB (NF-κB), and Wnt/β-catenin (Figure 3). Consequently, as will be presented in this review, Klotho exerts major effects on several biological processes relevant to aging and disease (Figure 4):

1) FGF23-dependent phosphate, calcium and vitamin D regulation.

In addition to these topics, we will report on several factors that increase Klotho, including commonly prescribed drugs, recombinant proteins, gene therapies, traditional medicines, nutraceuticals, and exercise.

Klotho is primarily produced in the kidney (renal tubules), but is also found in the brain (choroid plexus, CSF and neurons), pancreatic β cells, blood vessels and skin (Lim K, et al., 2015). Recent work also documents expression in peripheral blood circulating cells (Martin-Nunez et al., 2022). It is also excreted into the urine. Studies in nephrectomized rodents (Hu et al., 2016), and mice with kidney-specific genetic deletion of Klotho (Lindberg et al., 2014), have revealed the kidney is the principal source of circulating Klotho. It is produced by the proximal and distal convoluted renal tubules, perhaps more in the distal tubules (Lim K, et al., 2015). However, some authors report similar amounts of the membrane-bound form at both locations (Andrukhova et al., 2012; Erben and Andrukhova, 2017). Interestingly, Klotho has different functions in the proximal and distal tubules (Erben and Andrukhova, 2017). In the proximal tubule, Klotho promotes a phosphaturic effect and inhibits vitamin D production, whereas in the distal tubule it enhances Ca²⁺ reabsorption. Conditional gene knockout of Klotho in the proximal tubule reproduced many of the features of systemic gene knockout, confirming the importance of proximal tubular functions (Takeshita et al., 2017).

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-hydroxylase in the proximal renal tubule, which inhibits synthesis of active vitamin D, denoted 1,25(OH)₂D₃ (calcitriol). Among other effects, vitamin D promotes phosphate and calcium absorption in the gut, and this will be reduced. Furthermore, in a Klotho-independent manner, FGF23 reduces secretion of parathyroid hormone (PTH), which additionally influences phosphate calcium balance (Ben-Dov et al., 2007).

A deficiency of either Klotho or FGF23 in mice results in excessive 1α-hydroxylase activity, overproduction of active vitamin D, and associated hyperphosphatemia and hypercalcemia. In Klotho-deficient mice, hypervitaminosis D and hyperphosphatemia both appear to contribute to the accelerated aging phenotype (Kuro-o 2010, 2018, 2019, 2021). However, a low phosphate diet ameliorates disease, even though vitamin D further increases, suggesting that the hyperphosphatemia is more important than vitamin D toxicity in the aging syndrome. Nevertheless, ablation of vitamin D responses attenuated the lesions of Klotho-deficient mice (Anour et al., 2012). It has been proposed that hypersaturation of phosphate and calcium ions in the blood leads to the formation of calciprotein particles (CPPs) (Kuro-o, 2021). The CPPs precipitate in tissues, and are thought to accelerate the pathologic changes associated with aging.

The most common clinical cause of Klotho deficiency is kidney failure, which can result from many acute or chronic conditions (Neyra et al., 2021). Rare human mutations causing a severe deficiency of either Klotho or FGF23 are associated with hyperphosphatemia beginning at an early age (Ito and Fukumoto, 2021). These are autosomal recessive diseases. The subjects develop massive calcification in soft tissues, blood vessels, and in multiple organs and anatomic sites throughout the body. There are bone and dental lesions. There is also evidence of systemic inflammation. Treatment includes low phosphate diet, phosphaturic drugs, anti-inflammatory drugs, and phosphate binding agents. This severe congenital deficiency of either functional Klotho or FGF23 is not comparable to the much less reduced Klotho expression seen in chronic diseases described in this review. Indeed, these are not associated with the massive calcification observed in congenital hyperphosphatemia.

The Klotho homologue denoted β-Klotho (KLB) is an obligatory coreceptor for FGF19 and FGF21, which are both eFGFs (Kilkenny and Rocheleau., 2016). KLB, unlike Klotho, does not appear to have endocrine functions on its own. FGF19 plays a major role in bile acid homeostasis and liver metabolism (Dolegowska et al., 2019; Guthrie et al., 2022). FGF21 is involved in regulating lipid metabolism, insulin secretion and glucose homeostasis (Andersen et al., 2015; Bondurant and Potthoff, 2018; Dolegowska et al., 2019). FGF21 is produced in the liver, pancreatic islets, exocrine pancreas, and other tissues. The liver appears responsible for most of the circulating FGF21. The major metabolic functions of FGF21 have received considerable attention, and it has been examined in clinical trials for the treatment of obesity and diabetes.

Interestingly, Klotho is depleted in the islets of diabetic patients (Lin and Sun, 2015a). Several investigators have reported depressed circulating Klotho levels in subjects with type 1 diabetes (T1D) (Keles et al., 2016; Tarhani et al., 2020; Zubkiewicz-Kucharska, 2021); and type 2 diabetes (T2D) especially when there is advanced disease (Nie et al., 2017; Fountoulakis et al., 2018; Zhang and Liu, 2018). Circulating levels of s-Klotho are depressed in db/db mice (T2D) (Takenaka et al., 2011) and diabetic NOD mice (T1D) (Prud’homme et al., 2020). There is evidence that Klotho plays an important role in glucose and lipid metabolism, as reviewed (Razzaque, 2012; Donate-Correa et al., 2016; Wan Q, et al., 2017; Landry et al., 2021a). Klotho-deficient mice display islet atrophy and reduced insulin production (Razzaque, 2012). Klotho increases β-cell expression of TRPV2 and enhances Ca²⁺ entry and the glucose-induced response (Lin and Sun, 2012).

FGFR1c is expressed by β cells, and its attenuation results in diabetes and reduced numbers of β cells (Hart et al., 2000). Both Klotho and KLB are expressed by islet cells, and this suggests these cells can respond to FGF23 (Klotho coreceptor) and FGF21 (KLB coreceptor). FGFR1c promotes signaling through several key pathways for β cells (Figure 2), e.g., PI3K/Akt (promotes cell survival), RAS-MAPK (promotes mitogenic response), PLC-PPAR-adiponectin (regulates glucose and lipid metabolism). In accord with this, Klotho administered by gene transfer in mice demonstrated major protective effects on β cells in T1D or T2D models (Lin and Sun, 2015a,b). Importantly, Klotho reduced β-cell apoptosis and increased the proliferation of these cells. A key pathologic finding in NOD mice is the development of insulitis. This first appears as a peri-islet mononuclear cell infiltrate (including T cells), followed by focal invasion of immune cells into the islets, and subsequently severe infiltration and β-cell loss. Klotho gene transfer reduced insulitis, suggesting an immunosuppressive and/or anti-inflammatory effect. In addition, recombinant Klotho improved renal disease and hypertension in db/db mice (Takenaka et al., 2019). The authors concluded that Klotho inhibited TGF-β and tumor necrosis factor (TNF) signaling, to decrease renal fibrosis.

Systemic γ-aminobutyric acid (GABA) treatment protects against type 1 diabetes (T1D) in mice (Tian et al., 2004; Soltani et al., 2011; Wan Y, et al., 2015; Wang et al., 2019). It induces mouse and human β-cell replication/regeneration, while reducing the apoptosis of these cells (Liu W, et al., 2017, 2021; Purwana et al., 2014; Sarnobat et al., 2022; Soltani et al., 2011; Tian et al., 2013; Untereiner et al., 2019). In terms of mechanisms, we found that GABA increases the production of Klotho, in a multiple low-dose streptozotocin (STZ) model of T1D (Prud’homme et al., 2017a). GABA therapy increased Klotho in the pancreatic β cells, kidneys and plasma. Similarly, in the case of human islets transplanted into immunodeficient mice, we observed that oral GABA treatment increased β-cell Klotho expression and replication, and diminished apoptosis (Liu W, et al., 2021). These effects were ameliorated by co-administration of a dipeptidyl peptidase 4 (DPP-4) inhibitor.

In autoimmune diabetes-prone NOD mice, the injection of s-Klotho protein increased pancreatic β-cell replication and the β-cell mass (Prud’homme et al., 2020). It also reduced insulitis, whereas a Klotho blocking antibody had the opposite effect. These findings were similar to those of others who employed gene transfer methods (Lin and Sun, 2015a,b). In vitro, Klotho stimulated human β-cell survival, replication and insulin secretion (Prud’homme et al., 2017a). It also inhibited NF-κB activation, which could explain the anti-apoptotic effect. Klotho might also protect β cells through antioxidant mechanisms that can be induced, for instance, by Nrf2 and FoxO (Figure 3). Note that although GABA has only been applied in preclinical diabetes, there are numerous GABAergic drugs in clinical use (Palma et al., 2017), to treat epilepsy and other diseases. Unlike systemically applied GABA that is blocked by the BBB, many of these drugs cross this barrier and this can produce multiple adverse effects. Other than GABA, it is unknown whether GABAergic drugs increase Klotho levels.

Paradoxically, Klotho deficient mice (KL ^kl/kl ) are hypoglycemic, despite having low insulin levels (Razzaque 2012). This likely 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 Klotho is an appropriate agent to treat diabetes. We hypothesize that Klotho therapy of diabetes will be helpful when normal plasma levels are restored. Interestingly, overexpression of Klotho in transgenic mice produced slight insulin resistance, and extended lifespan (Kurosu et al., 2005). In experimental T1D and T2D, as outlined in this review, Klotho therapy was beneficial. Whether humans will respond similarly is unknown. In our work, Klotho protein was administered at a low dose, every 48 h (Prud’homme et al., 2020). It has a half-life of only 7 h (Hu et al., 2016), which is relevant to efficacy and potential adverse effects.

One key issue regarding antidiabetic drugs such as GABA and GLP-1 is whether their general antidiabetic effects (Wang Q, et al., 2019; Drucker, 2018) enhance Klotho indirectly, or there is a more direct effect on Klotho gene expression. This has not been extensively studied, but we hypothesize this is due to a combination of mechanisms. In STZ-induced diabetes, STZ is toxic to the kidney (Cheng MF et al., 2010), not just pancreatic β cells, and it reduces Klotho in the circulation and kidneys (Prud’homme et al., 2017a). This was almost completely reversed by oral GABA administration. Studies of others have shown that GABA treatment protects the kidneys against tubular fibrosis and atrophy, in the subtotal nephrectomy model (Sasaki et al., 2007), and ischemia-reperfusion injury (Kobuchi et al., 2009). This is consistent with the fact that renal tubular cells express GABA receptors (Amenta et al., 1988; Sarang et al., 2008; Takano et al., 2014). Thus, GABA may be protecting renal tubular cells against injury, such that they continue producing Klotho at near-normal levels. However, GABA and/or GLP-1RA directly induced Klotho expression in pancreatic β cells. Of interest, the Klotho promoter has a transcription binding site for PAX4, which is a transcription factor essential for the differentiation of β cells in the pancreas (Sosa-Pineda et al., 1997; Xu and Sun, 2015). GLP-1 induces the expression of PAX4 in human islets (Brun et al., 2008), and therefore, this may (along with other factors) induce the expression of Klotho.

In addition to diabetes, Klotho has been found to have role in obesity. In humans, CSF Klotho levels are depressed in obese subjects (Landry et al., 2021a,b). Intracerebroventricular injection of Klotho in diabetes-prone mice reduced food intake, ameliorated glucose homeostasis and reduced body weight (Landry et al., 2020). This was related to FGFR signaling in neurons. Studies by these authors showed that Klotho acts on the arcuate nucleus of the hypothalamus (ARC) to regulate metabolism (Landry et al., 2021b). Neurons and astrocytes were both targeted, and FGFR signaling through PI3K (neurons) or ERK (astrocytes) was documented. Interestingly, they found no correlation between systemic and CSF concentrations of Klotho, and the mechanism of Klotho regulation in the CSF remains unclear. Circulating Klotho also appears to play and important role in metabolism. Systemic Klotho treatment in mice reduced adiposity and lipid accumulation in the liver; and increased lean mass and energy expenditure (Rao Z, et al., 2019). With Klotho treatment, quantitative PCR revealed lower expression of lipogenic genes.

There is considerable evidence that Klotho is a tumor suppressor molecule, as reviewed elsewhere (Rubinek and Wolf, 2016; Abolghasemi et al., 2019; Sachdeva et al., 2020; Ewendt et al., 2021). This is consistent with its capacity to inhibit pathways linked to cancer, including Wnt, IGF-1, TGF-β and NF-κB, as outlined previously (Figure 3; Table 1). The expression of Klotho is depressed or silenced in almost all types of cancer examined. Klotho-positive tumors generally have a better prognosis. The suppression of Klotho appears to occur by the same mechanism as other tumor suppressor genes. For instance, this involves DNA hypermethylation at promoter sites, histone modifications and miRNAs (Rubinek and Wolf, 2016; Abolghasemi et al., 2019). In experimental tumor models, Klotho has anti-tumor effects in vivo and/or in vitro. This was demonstrated in breast cancer (Wolf et al., 2008; Ligumsky et al., 2015), pancreatic cancer (Abramovitz et al., 2011), and other cancers including colon cancer, gastric cancer, and hepatocellular carcinoma (HCC). For instance, in pancreatic cancer, Klotho was poorly expressed in the tumor (Abramovitz et al., 2011). Overexpression of Klotho, or therapy with s-Klotho, inhibited the growth of pancreatic cancer cells. Indeed, multiple studies have shown that Klotho inhibits cancer-cell proliferation, colony formation and invasion; and promotes apoptosis and autophagy (Sachdeva et al., 2020). Similarly, in vivo, forced tumor overexpression of Klotho, or systemic administration of s-Klotho, inhibited the growth of several types of tumors. Importantly, this includes human tumors xenografted into mice. The membrane-bound and soluble forms of Klotho both have tumor suppressor effects.

Low Klotho levels correlate with neurodegenerative disease and cognitive impairment (Vo et al., 2018; Hanson et al., 2021), as well as frailty (Veronesi et al., 2021). For example, in nursing home residents, low serum Klotho was associated with poor cognition, frailty, dependence, and frequent falls (Sanz et al., 2021). The role of Klotho in the brain is not well understood, but there is evidence it is neuroprotective. In the central nervous system, Klotho is produced by the choroid plexus, and is found in the CSF. It is also expressed widely throughout the brain, predominantly in grey matter areas including the hippocampus (Clinton et al., 2013; Cararo-Lopes et al., 2017). It is produced by both neurons and oligodendrocytes. Klotho expression in the brain is decreased in aging subjects and early Alzheimer’s disease (Fung et al., 2022). In mouse models of Alzheimer’s disease, Klotho overexpression is beneficial.

Morphologically, in Alzheimer’s disease, the brain is characterized by an excess of β-amyloid (Aβ) plaques, as well as neuronal aggregation of Tau protein forming neurofibrillary 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 is thought to protect against the toxicity of Aβ and NFTs (Zeldich et al., 2014; Fung et al., 2022). Aβ is generated from the proteolytic cleavage of amyloid precursor protein (APP) by secretases (Lichtenthaler et al., 2022). APP is a type 1 transmembrane protein, and its cleavage by an α-secretase results in the release (shedding) of a large extracellular segment, which does not form amyloid. This cleavage process is accomplished primarily by ADAM10, and is similar to the generation of s-Klotho. However, processing of APP by β-secretase and γ-secretase generates small secreted Aβ peptides that form amyloid. Interestingly, α-secretase cleavage prevents the subsequent generation of amyloidogenic peptides (Lichtenthaler et al., 2022). Therefore, increasing α-secretase activity, or decreasing β- and γ-secretase activity, might be of benefit.

One potential mechanism of disease involves deficient autophagy, which is a process that normally degrades and removes proteins and other substances, preventing their toxic accumulation in the cell. Klotho ameliorates autophagy, and this might protect against neurodegenerative conditions like Alzheimer’s (Fung et al., 2022). Klotho also appears to contribute to oligodendrocytic myelin production, and the maintenance of white matter integrity. There is evidence of an inflammatory component to Alzheimer’s disease, as well as some other neurodegenerative diseases, possibly involving NF-κB activation (Ju wang et al., 2019). Thus, as in other sites, Klotho may exert beneficial anti-inflammatory and anti-oxidative actions in the brain. Klotho might promote differentiation of microglia to the anti-inflammatory type (M2), instead of the inflammatory type (M1) (Fung et al., 2022).

The hippocampus plays a major role in memory. It is one of the few regions of the brain that sustains neurogenesis in adulthood, and there is loss of neurons in that area in Alzheimer’s disease (Rao YL, et al., 2022). Remarkably, recent work indicates that Klotho stimulates neurogenesis in the hippocampus. This was evident, for example, when comparing mice with Klotho deficiency vs. those overexpressing Klotho (Laszczyk et al., 2017). In accord with neuronal loss, Klotho deficient mice demonstrate severe cognitive defects. Recently, intermittent fasting in mice was shown to increase Klotho expression and neurogenesis in the hippocampus (Dias et al., 2021). Intermittent fasting proved superior to caloric restriction, and it was associated with improvement of long-term memory. Klotho-deficient mice did not respond to intermittent fasting. It is possible that drug therapy can achieve similar results. For instance, in a rat model of metabolic syndrome, the DPP-4 inhibitor vildagliptin (increases GLP-1) augmented hippocampal Klotho, concurrently decreased inflammatory and apoptotic biomarkers at that site, and prevented neurodegenerative changes (Yossef et al., 2020). This improved memory in treated rats. Note that most DPP-4 inhibitors, including vildagliptin, do not cross the blood-brain barrier (BBB) but they enhance circulating GLP-1 (Deacon, 2019), which does cross. In a rat model of STZ-induced cognitive impairment, simvastatin increased hippocampal Klotho and improved cognitive function (Adeli et al., 2017). Similarly, others found that rosiglitazone treatment improved cerebral Klotho expression (Chen LJ, et al., 2014). In the latter study, intracerebroventricular infusion of recombinant Klotho was also beneficial. Alternatively, some authors have delivered Klotho by gene transfer (Table 2), such as intracerebroventricular injection of a lentiviral vector encoding Klotho (Zeng et al., 2019). The findings outlined above regarding Klotho in neurodegenerative pathologies have all been obtained in rodents, and it remains to be determined whether they are applicable to humans.

To our knowledge, Klotho-based therapy has not been applied in humans with neurodegenerative diseases. Nevertheless, compared to subjects with low levels of Klotho, those with high levels (CSF or plasma) have superior cognitive functions, and a 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 recently reported that serum and CSF levels correlate strongly, and 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 caveat is that the reasons for discrepancies between studies are not clear, concerning Klotho levels in the CSF or the circulation. There has been much concern about the specificity of some anti-Klotho antibodies used in ELISA and other assays (Kuro-o, 2019). Some authors recently examined this question in human serum samples from patients with a wide range of kidney function (Neyra et al., 2020). This was done with a widely used commercial ELISA, as well as immunoprecipitation-immunoblotting (IP-IB) with a highly specific Klotho antibody. The IP-IB Klotho results correlated with kidney function much better than the ELISA. Freeze-thaw cycles impaired Klotho detection. These findings suggest that relying solely on an ELISA assay could be misleading, and sample handling is important. Another relevant observation is the wide variability in Klotho levels between individuals. For instance, in a 18–35 age group approximately half of individuals had low levels, comparable to aged subjects, whereas the other half had high levels (Espuch-Oliver et al., 2022). In the 55–85 age group, however, levels were more consistently low. CSF levels are also quite variable (Kundu et al., 2022). In view of this, results obtained from small groups should be interpreted with caution. The biological significance of this variability is unclear, and long-term studies are required.

A Klotho gene variant denoted KL-VS (2 amino acid substitutions in the protein) appears to provide some protection against Alzheimer’s disease, but only in the heterozygous state (Driscoll et al., 2021; Nietzel et al., 2021). Beneficial effects were only observed in some subgroups that were studied. The mode of action of this variant is unclear although, interestingly, a reduction of Tau accumulation has been reported.

In this manuscript we explored the many facets of Klotho biology. The best characterized aspects relate to renal physiology, and especially phosphate and calcium homeostasis. The importance of the FGF23/Klotho/FGFR interaction was evident in Klotho deficient mice. The major outcomes were hyperphosphatemia and hypervitaminosis D. The accelerated aging syndrome of these mice was greatly ameliorated by a low phosphate diet. Rare mutations of either Klotho or FGF23 in humans result in a hyperphosphatemic syndrome, characterized by massive tissue calcification and systemic inflammation. In chronic renal disease of various etiology, as well as diabetes, Klotho production is decreased. It has been proposed that declining renal function, and associated low Klotho, accelerate or aggravate many of the pathologies found in old age. This may involve, for example, the precipitation of CPPs in tissue (Kuro-o, 2021).

It seems unlikely that all the manifestations of Klotho insufficiency are related to FGF23-induced signaling. Indeed, as outlined in several sections, Klotho blocks major pathways that play a role in aging. This refers specifically to inhibition of TGF-β, Wnt, IGF-1 and NF-κB. These are complex pathways that impact on a vast number of biological processes. Thus, regardless of other mechanisms that may drive aging, the overactivation of these pathways is likely to be important. This is most evident in fibrosis, inflammation and cancer, which are all countered by Klotho. Furthermore, Klotho activates antioxidant pathways, such as Nrf2 and FOXOs, which are thought to mediate antiaging effects.

In terms of future directions, there are several important avenues. Klotho is described as an antiaging molecule, although there is no direct evidence that it delays aging in humans. This is one of the most important questions to address, but it will require very long-term studies. Klotho can be a marker of disease (kidney, brain or other), or a therapeutic molecule. In particular, the function(s) of Klotho in the brain are not well defined, but neuroprotective effects are apparent. Klotho levels in the circulation or CSF could indicate susceptibility to disease. Multiple drugs appear to increase Klotho, but studies in humans are scant. Does Klotho contribute to the therapeutic effects of some drugs used to treat hypertension, hyperlipidemia, diabetes or transplant rejection? Could some of these drugs ameliorate neurodegenerative diseases by enhancing Klotho? This is all unknown. Indeed, there have been practically no clinical investigations of Klotho-related treatment, outside of preclinical work. Another important aspect is the role of Klotho as a tumor suppressor molecule. This is well documented in animals models, but needs to be developed in the clinical context. The slim clinical applications of Klotho research can perhaps be explained by the limited understanding of its mechanisms of action, especially outside the kidney. However, knowledge of the molecular and physiological aspects of Klotho has progressed considerably in recent years, as outlined in this review.

For therapy, several approaches are feasible, as described in this manuscript. Clinical research focused on any of these approaches would be an important contribution to the field. The most direct approach is the administration of recombinant Klotho or peptides. In view of the relatively short half-life of these products, there is a need to produce long-acting constructs, or slow-release formulations. There is little evidence from preclinical work that Klotho administration is toxic, at least when restoring normal 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, there are also commonly available over-the-counter medicines, supplements, or nutraceuticals that increase Klotho. This is based on animal work, and it would be of much interest to examine the possibility of Klotho enhancement in humans. In the case of neurodegenerative diseases it is relevant that some drugs, including some traditional medicines, cross the BBB and might increase Klotho in the brain. Finally, exercise and the long-term maintenance of fitness are simple methods to increase Klotho. In conclusion, most of the studies on Klotho have been performed in 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 favor the clinical investigation of Klotho-based treatments.