Regulation of αKlotho

Keywords

αKlotho FGF23 CKD Longevity

Abstract

Since its discovery in 1997, αKlotho has gained a lot of attention due to its powerful anti-aging and health-promoting properties. It exists as a membrane-bound protein or as a soluble factor. Membrane-bound αKlotho is an essential cofactor for fibroblast growth factor 23 (FGF23), thereby being involved in the regulation of renal phosphate and vitamin D metabolism. Soluble αKlotho (sKL) is present in different body fluids and exerts hormone-like effects. Through the αKlotho-FGF23 signaling axis, FGF23 regulates phosphate excretion by downregulating Na⁺-dependent phosphate transporter (NaPi-2a). In addition, this axis suppresses expression of 1α-hydroxylase, thereby reducing active vitamin D (calcitriol) serum concentration. Disruptions of this axis lead to deranged mineral metabolism. Low levels of αKlotho and elevated FGF23 are early biomarkers for different diseases, including chronic kidney disease (CKD) and cardiovascular diseases (CVD). In CKD, decreased renal αKlotho expression and enhanced FGF23 production contribute to worsening kidney function. Activated transforming growth factor b1 (TGF-b1) signaling, promoting renal fibrosis, contributes to the pathophysiology. Moreover, FGF23 directly induces left ventricular hypertrophy (LVH) through FGF receptor-induced calcineurin/nuclear factor of activated T cells (NFAT) signaling in CKD. Our review aims to comprehensively summarize the regulation and function of αKlotho, highlighting its central role in maintaining mineral metabolism and its therapeutic potential in age-related and chronic diseases.

Introduction

The anti-aging protein αKlotho owes its name to Greek mythology, in which the goddess Klotho decided over life and death and held the threads of life [1, 2]. Its discovery goes back to experiments with a kl/kl mouse strain in 1997 [1]. This mouse strain is characterized by changes in behavior and appearance at a few weeks of age only [1]. Particularly striking is a drastic loss of bone mineral density and further signs of premature aging, leading to early death [1]. Conversely, overexpression of αKlotho delays aging and induces longevity, making αKlotho an interesting target in longevity research [3].

Klotho family

Three different Klotho proteins exist, termed α-, β- and γ-Klotho, all being expressed in different organs and fulfilling various functions [4], but this review focuses only on αKlotho.

The latter is strongly expressed in the brain and kidney, and to a much lesser extent in the pituitary gland, aorta or pancreas [1]. αKlotho belongs to the group of type I membrane proteins with several structural domains: two extracellular domains KL1 and KL2, a transmembrane domain (TM) and a short cytoplasmic site (CYT) [5–7]. Depending on the cleavage site, membrane-bound αKlotho protein can be split into full-length soluble αKlotho (sKL) or into the respective single fragments KL1 and KL2 by a disintegrin and metalloproteinase (ADAM)10 or 17 [8–10]. In addition, a product of alternative RNA splicing exists, namely secreted αKlotho and identical to KL1 [5, 10]. Both human and mouse transcripts of membrane αKlotho comprise five exons, whereas the human secreted form of αKlotho consists of five and mouse secreted αKlotho only consists of three exons [5, 11]. Secreted αKlotho transcripts can only be detected in mice and humans, but not in rats [12] and the expression of secreted αKlotho in humans is even higher than that of membrane αKlotho [5].

In contrast to soluble and secreted αKlotho (summarized as circulating KL) [13] being humoral factors [14], the function of the membrane-bound form is much better understood: It acts as an essential cofactor for the binding of fibroblast growth factor 23 (FGF23) to its receptor since only αKlotho generates a specific FGF23 receptor FGFR) complex FGFR1c, FGFR3c or FGFR4 [15, 16].

Membrane-bound αKlotho and FGF23

FGF23 was first described in 2000 [17] and is predominantly expressed by bone cells, i.e. osteoblasts and osteocytes [18]. The discovery of missense mutations in the FGF23 gene accounting for derangements of phosphate metabolism, rickets and further disorders of bone, led to the assumption that FGF23 is a major factor for phosphate and vitamin D metabolism [17, 19].

Altogether, 22 FGF genes exist that can be divided into intracellular and secreted FGFs, the latter having paracrine and endocrine functions [20, 21] and comprising FGF15/FGF19, FGF21, and FGF23 [22]. In contrast to the other FGF subfamilies, endocrine FGFs only have low affinity for heparin, resulting in a weak FGF receptor interaction [21–23]. It is the primary task of αKlotho to facilitate efficient and specific FGF23 signaling in the kidney by forming a FGFR1(IIIc)-αKlotho complex (Fig. 1) [16, 24]. It controls calcitriol synthesis by regulating the expression of its key enzyme, 1α-hydroxylase, in the proximal tubule [25, 26]. By downregulating the major renal Na+-dependent phosphate transporter NaPi-2a, FGF23 suppresses phosphate reabsorption [26]. Both, αKlotho or FGF23 deficiency result in similar disorders in mice that are mainly due to deranged vitamin D and phosphate homeostasis and further characterized by growth retardation and a severely reduced life span [4, 24]. Moreover, αKlotho and FGF23 may both serve as biomarkers for the early detection of various diseases. Particularly in chronic kidney disease (CKD), an early rise in FGF23 serum levels as well as a decrease αKlotho serum levels are predictors of CKD progression [27, 28]. In an αKlotho-independent manner, elevated FGF23 binds to FGFR4 on cardiomyocytes and thereby activates phospholipase Cγ (PLCγ)/calcineurin/nuclear factor of activated T cells (NFAT) signaling, inducing left ventricular hypertrophy [29, 30].

Soluble αKlotho (sKL)

As the product of cleaved renal membrane-bound αKlotho, sKL serves as a hormone-like factor independently of FGF23 [31]. It can be detected in blood, cerebrospinal fluid, or urine [31] and is effective in different organs, including heart and blood vessels [32]. SKL has organoprotective properties in the heart by reducing susceptibility to stress signals and lowering intracellular calcium levels by inhibition of transient receptor potential channel TRPC6 [33]. It has beneficial effects in blood vessels by reducing vascular calcification [34, 35] and is nephroprotective [36]. SKL controls important intracellular signaling pathways including transforming growth factor-β (TGF-β) or Wnt signaling [31]. Antitumor [37] or antifibrosis effects of sKL may also be due to TGF-β receptor or Wnt signaling inhibition [38, 39]

Hitherto, no receptor for sKL has been characterized, but sKL binds to so-called lipid rafts and thereby negatively affects phosphoinositide 3-kinase (PI3K) signaling [40]. Lipid rafts are considered a promising target for many sKL-induced pathways [40].

Regulation of αKlotho in the kidney

Regulators of renal αKlotho expression are reviewed below and listed in an alphabetical order (summarized in Table 1).

1, 25-dihydroxyvitamin D3
In cell lines of proximal or distal tubular origin or of the collecting duct, 1, 25-dihydroxyvitamin D3 (1, 25D) enhances renal αKlotho gene expression, an effect dependent on vitamin D receptor (VDR) [41, 42].
Also, the administration of 1, 25D is paralleled by an increase in αKlotho gene expression in mice [43].

Albumin
Albumin reduces αKlotho mRNA and protein abundance in vitro and in vivo [44, 45], an effect attributed to albumin-induced endoplasmic reticulum (ER) stress. Conversely, inhibition of ER stress or silencing of activating transcription factor 3 (ATF3) enhance αKlotho protein [44].

AMP-dependent kinase
AMP-dependent kinase (AMPK) is activated in cellular states of energy deficiency characterized by high levels of AMP [46]. It stimulates renal αKlotho gene and protein expression in vitro [47], but αKlotho itself can activate AMPK signaling, too [48].

Cytotoxic agents
In certain renal cell lines, αKlotho expression is enhanced by cisplatin, paclitaxel, or doxorubicin [49], an effect at least in part involving peroxisome proliferator-activated receptor γ (PPARγ) [49]. The induction of apoptosis with PAC-1 shows a similar effect on αKlotho expression in vitro [49]. In contrast, these cytotoxic drugs suppress renal αKlotho gene expression and reduce sKL in human kidney 2 (HK2) cells [49].

D-galactose
D-galactose stimulates renal fibrosis by inducing silent mating type information regulation 2 homolog-2 (SIRT2) and TGF-β1, an effect paralleled by reduced renal αKlotho protein abundance in vivo [50]. Conversely, SIRT2 inhibitor acylglycerol kinase (AGK)-2 upregulates αKlotho protein [50].

Epidermal growth factor
Epidermal growth factor (EGF) elevates renal αKlotho mRNA levels in vitro [51].

Erythropoietin
Recombinant human erythropoietin (EPO) induces renal αKlotho protein expression in rats with acute nephropathy [52].

Histone deacetylase 3
Histone deacetylase (HDAC) inhibition up-regulates αKlotho mRNA and protein in a kidney cell line or in vivo and delays CKD progression [53, 54]. HDAC3 is a regulator of ROS production and is involved in renal fibrosis [55]. TGF-β activates HDAC3 that subsequently decreases αKlotho protein [56]. In contrast, inhibition of HDAC3 stimulates both αKlotho gene and protein expression in vitro, while increased αKlotho protein expression is reported in vivo [56].

Inflammation
Lipopolysaccharides (LPS) downregulate renal αKlotho gene expression [12] and protein [57] in vivo and in vitro. Also, tumor necrosis factor α (TNFα) and TNF-like weak inducer of apoptosis (TWEAK) suppress αKlotho mRNA and protein expression through NFκB signaling in vitro and in vivo [58], as does interferon (IFN)-γ in vitro [59].

Metabolic factors
High levels of glucose, especially in type 2 diabetes, are negatively associated with αKlotho mRNA and protein abundance in a proximal tubular cell line [60].
PPARγ agonists including troglitazone upregulate renal αKlotho gene and protein expression in vitro and in vivo [61, 62]

Klotho-derived peptide 1
Klotho-derived peptide 1 (KP1), an inhibitor of TGF-β1 signaling pathway as a ligand of TGF-β receptor 2, is positively associated with αKlotho protein expression in vitro and in vivo [63, 64].

Lithium
Lithium reduces renal αKlotho protein abundance in vivo [65].

Nutrition and lifestyle
Berberine, a natural plant compound has anti-inflammatory, anti-oxidative and anti-apoptotic properties [66]. In acute kidney injury, it upregulates renal αKlotho gene expression [66].
Resveratrol, a polyphenol available in many plant-based foods, stimulates renal αKlotho gene and protein expression in vitro and in vivo [67, 68].
A high phosphate diet suppresses renal αKlotho protein abundance in wild type mice [69]. Its impact is stronger in adolescent mice compared to adult animals [70].
Nicotinamide attenuates the decrease of αKlotho protein expression in mice with glycerol-induced AKI by altering NFκB and histone deacetylase 1 activity [71].
Aerobic exercise elevates renal αKlotho gene and protein expression and reduces ROS production [72] as well as TGF-β1 signaling [73].

PKC
Protein kinase C (PKC), especially isoform PKCγ, activation downregulates αKlotho gene expression in vitro [74].

Rapamycin
Rapamycin is an mTOR (molecular target of rapamycin) inhibitor [75]. One study found upregulation of renal αKlotho protein in mice by rapamycin [75] whereas another one reported rapamycin-induced downregulation of αKlotho transcripts and protein abundance in rats [76].

Renin-angiotensin system
Water homeostasis controls renal αKlotho expression. Dehydration induces angiotensin II, an effect paralleled by suppression of αKlotho mRNA and protein levels [77]. Angiotensin II is a direct negative regulator of αKlotho gene and protein expression in vitro [78].
In vitro or in vivo, vasopressin [77] and aldosterone [77, 79] reduce the expression of renal αKlotho gene and protein, while aldosterone antagonist spironolactone induces it [80]. Both losartan (angiotensin II receptor antagonist [81]) and fosinopril (inhibitor of angiotensin-converting enzyme (ACE) [82]), enhance renal αKlotho gene and protein expression in a mouse model of primary hypertension [83].

Reactive oxygen species
Reactive oxygen species (ROS) are negative regulators of renal αKlotho gene and protein expression in vitro [84, 85] with nuclear factor erythroid 2-related factor 2 (Nrf2) being involved [85].

Sodium-glucose co-transporter-2 inhibitors
Sodium-glucose co-transporter-2 inhibitors (SGLT2i) canagliflozin, dapagliflozin, empagliflozin or sotagliflozin are reported to exert contrasting effects on αKlotho gene and protein expression in different renal cell lines and attenuate the decrease of αKlotho triggered by albuminuria or inflammation [60, 86].

Statins
Statins upregulate renal αKlotho mRNA and protein expression in vitro and in vivo [87–89]. The upregulation is dependent on inhibition of RhoA pathway [88].

Toxins
αKlotho gene and protein expression is downregulated in the presence of uremic toxin indoxyl sulfate in vitro and in vivo [90, 91]. AST-120, an adsorbent of indole, reverses the suppressive effect on αKlotho protein [91, 92].
Shiga toxin 2 downregulates renal αKlotho mRNA and protein abundance in mice [93].

Transcription factor Sp1
Overexpression of the ubiquitously expressed transcription factor Sp1 upregulates αKlotho transcripts and protein in vitro [94].

Regulation of αKlotho in organs and tissues other than kidney

Regulators of αKlotho expression in extrarenal organs or tissues are reviewed below and listed in alphabetical order (summarized in Table 2).

Aerobic exercise
Aerobic exercise upregulates αKlotho gene and protein expression in rat brain [72].

Cadmium
Cadmium exposure is negatively associated with αKlotho protein expression in rat hippocampus and in a cell line derived from the adrenal gland [95].

Calcitonin gene-related peptide
In endothelial progenitor cells, calcitonin gene-related peptide (CGRP) upregulates αKlotho gene and protein expression and reverses angiotensin II-induced senescence [96].

Estradiol
αKlotho protein is enhanced by estradiol E2 in rat hippocampus, an effect related to cognitive function and synapse formation [97].

Histone deacetylase inhibition
Inhibition of HDAC elevates αKlotho mRNA levels in femurs of mice [54].

Matrix stiffness
Matrix stiffness, a typical feature of aging, is implicated in decreased αKlotho expression in chondrocytes, and abolishment of stiffness enhances αKlotho expression in vivo [98].

Rapamycin
In addition to the kidney, αKlotho protein is also upregulated by rapamycin in adipose tissue, lung, muscle, brain and heart [75].
Rapamycin also increases αKlotho mRNA and protein levels in some cell lines derived from the aorta or in the aorta of mice or rats and thus counteracts vascular calcification [99].

Resveratrol
Treatment with resveratrol elevates αKlotho gene and protein abundance in mouse brain dose-dependently [67].

Triiodothyronine
Triiodothyronine (T3) increases mRNA levels of the membrane form of αKlotho in preadipocytes during differentiation [100].

Regulation of sKL and secreted αKlotho

Regulators of sKL or secreted αKlotho expression are reviewed below and listed in alphabetical order (summarized in Table 3).

1, 25-dihydroxyvitamin D3
In a cell line of distal tubular origin or of the collecting duct, 1,25-dihydroxyvitamin D3 (1, 25D) enhances mRNA levels of secreted αKlotho identified with a primer pair that specifically amplifies the secreted αKlotho splice form [41].
An increase in serum and urinary αKlotho also occurs in mice with CKD on a high phosphate diet treated with vitamin D receptor agonists [101]. In contrast, cholecalciferol does not significantly change sKL [102] or even reduces it [103] in patients on dialysis.

Albumin
Albumin reduces secreted αKlotho mRNA expression in vivo [44]. Furthermore, αKlotho protein levels are reduced in the urine of patients with renal dysfunction as a consequence of severe albuminuria [45].

Calcimimetics
The calcium-sensing receptor CaSR activates ADAM10 in the kidney, thereby being involved in Klotho shedding [104]. SKL is elevated upon treatment with calcimimetics or alkali in vitro and in vivo [104], an effect dependent on CaSR, ADAM10, and tetraspanin 5 [104, 105].

Histone deacetylase inhibition
Inhibition of HDAC elevates αKlotho protein levels in mouse serum [54].

Hormones
According to a human study, the sKL serum concentration is positively correlated with total and free triiodothyronine (T3) [106]. T3 increases αKlotho but not sKL gene expression in a preadipocyte cell line during differentiation [100]. In patients with hyper- or hypothyroidism sKL protein is reduced [107].

Hypertension
Elevated blood pressure lowers serum sKL levels [108].

KP1
KP1 increases sKL protein levels in mice with fibrotic kidney [64].

Lifestyle
Aerobic exercise is associated with elevated sKL plasma levels in a human study [109] as is adequate sleep [110].

Metabolic factors
Insulin elevates sKL by enhancing ADAM10- and ADAM17-mediated shedding of transmembrane Klotho [8]. SKL and secreted αKlotho are downregulated by adiponectin in vivo and in vitro [111].
In mice treated with streptozotocin that induces type 1 diabetes, gamma-aminobutyric acid (GABA) enhances sKL[112].

Rapamycin
In rats with CKD, rapamycin elevates serum sKL levels [99].

SGLT2 inhibitors
In patients treated with SGLT2 inhibitors for type 2 diabetes, sKL in serum and urine is upregulated [60].

Administration of exogenous Klotho as therapeutic agent

The administration of exogenous Klotho protein may be a promising approach in the treatment of different diseases. Exogenous Klotho may be comparable to sKL and may thus provide resistance of cells to oxidative stress via inhibition of the insulin/PI3K/Akt signaling pathway and FoxO-mediated upregulation of anti-oxidative enzymes [113, 114]. Moreover, sKL not only ameliorates renal fibrosis and CKD [115, 116], but also acts as a tumor suppressor in various types of cancer [117–119]. Further health-promoting effects of exogenous Klotho administration are part of current research and already reviewed elsewhere [120].

Conclusion

αKlotho in both of its form (membrane-bound or soluble) is an important regulator of health and disease. Due to its anti-aging effects, αKlotho has gained attention as a putative therapeutic target. It not only preserves kidney function, but also positively affects the heart, blood vessels or cognitive functions and improves outcomes in cancer or diabetes. As summarized in this article, regulation of αKlotho is complex and dependent on several factors. For sure, more research is needed to better understand the physiological and pathophysiological roles of membrane-bound αKlotho and sKL.

Acknowledgements

Fig. 1 was partly generated using Servier Medical Art (https://smart.servier.com/), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). No AI was applied.

Author Contributions
Julia Vogt and Michael Föller wrote the paper.

Funding Sources
The author’s research into regulation of αKlotho was supported by Deutsche Forschungsgemeinschaft.

Statement of Ethics
The authors have no ethical conflicts to disclose.

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