Abstract
The discovery of the Klotho (KL) gene, which was originally identified as a putative aging-suppressor gene, has generated tremendous interest and has advanced understanding of the aging process. In mice, the overexpression of the KL gene extends the life span, whereas mutations to the KL gene shorten the life span. The human KL gene encodes the α-Klotho protein, which is a multifunctional protein that regulates the metabolism of phosphate, calcium, and vitamin D. α-Klotho also may function as a hormone, although the α-Klotho receptor(s) has not been found. Point mutations of the KL gene in humans are associated with hypertension and kidney disease, which suggests that α-Klotho may be essential to the maintenance of normal renal function. Three α-Klotho protein types with potentially different functions have been identified: a full-length transmembrane α-Klotho, a truncated soluble α-Klotho, and a secreted α-Klotho. Recent evidence suggests that α-Klotho suppresses the insulin and Wnt signaling pathways, inhibits oxidative stress, and regulates phosphatase and calcium absorption. In this review, we provide an update on recent advances in the understanding of the molecular, genetic, biochemical, and physiological properties of the KL gene. Specifically, this review focuses on the structure of the KL gene and the factors that regulate KL gene transcription, the key sites in the regulation of α-Klotho enzyme activity, the α-Klotho signaling pathways, and the molecular mechanisms that underlie α-Klotho function. This current understanding of the molecular biology of the α-Klotho protein may offer new insights into its function and role in aging.
Introduction
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Klotho Gene
KL gene localization
KL promoter characteristics
KL gene structure
Other KL gene properties
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α-Klotho Transcription and Post-translational Modification
Transcription
Post-translational modification
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α-Klotho Protein Characteristics
α-Klotho size, sequence, and activity
α-Klotho H/R mutation
α-Klotho KL-VS mutation
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Transmembrane and Soluble α-Klotho Functions
Inhibition of the insulin/IGF-1 signaling pathway
Suppression of WNT signaling
Participation in Ca2+ homeostasis
Participation in phosphate homeostasis
Suppression of oxidative stress
Other functions of Klotho
Circulating α-Klotho Function
Summary and Perspectives
I. Introduction
In Greek mythology, Klotho (Clotho), one of the Moirai, spins the thread of life and controls the ultimate destiny of humans. The aging-suppressor gene that was identified by Kuro-o et al (1) in 1997 was named Klotho. An insertion mutation in the 5′ flanking region of the α-Klotho gene in mice was associated with several symptoms of premature aging, including soft tissue calcification, arteriosclerosis, skin atrophy, gonadal dysplasia, infertility, hypoglycemia, severe hyperphosphatemia, osteoporosis, emphysema, and an overall shorter life span (1). Conversely, the overexpression of the mouse α-Klotho gene slowed the aging process and extended the life span by 20–30% (1–3). Together these results indicate that α-Klotho may play a critical role in the regulation of the aging process (4). The α-Klotho gene is highly conserved in humans, mice, and rats and is also found in Danio rerio and Caenorhabditis elegans (4). The α-Klotho protein is also highly homologous across species (Table 1). In particular, the α-Klotho protein sequence is 98% identical between humans and mice (Table 1).
Table 1.
Basic Information of Klotho in Humans, Mice, and Rats
Species | Name | Protein | mRNA | Genomic | Protein Identity, % |
---|---|---|---|---|---|
Homo sapiens | klotho | NP_004786.2 | NM_004795.3 | NG_011485.1 | 100 |
Mus musculus | klotho | NP_038851.2 | NM_013823.2 | NC_000071.6 | 98 |
Rattus norvegicus | klotho | NP_112626.1 | NM_031336.1 | NC_005111.3 | 86 |
Canis lupus familiaris | klotho | XM_003433309.1 | NC_006607.3 | 88 |
The discovery of the α-Klotho gene has generated tremendous research interest that has significantly advanced our current understanding of the aging process. In humans, serum levels of α-Klotho decrease with age after age 40 years (5–7). This decrease in α-Klotho levels may be observed in patients with several aging-related diseases such as cancer, hypertension, and kidney disease (4, 8–11). A clinical study demonstrated that the G395A single-nucleotide polymorphism (SNP) in the promoter region of the α-Klotho gene was associated with essential hypertension, especially in subjects over 60 years old (8). This study also found, however, that the A variant of the G395A SNP, which is associated with higher promoter activity, may be protective against the development of essential hypertension by upregulating α-Klotho gene expression (8). Klotho deficiency, as in KL mutant heterozygous (+/−) mice, results in salt-sensitive hypertension (12). In vivo expression of the α-Klotho gene was shown to prevent the progression of hypertension and attenuate kidney damage in spontaneous hypertensive rats (9). Klotho deficiency also is associated with human chronic renal failure (13), and it promotes early diabetic nephropathy in mice (14). Recent studies showed that Klotho is also expressed in pancreatic β-cells, promotes insulin release, and protects β-cells in type II diabetes (15, 16).
Although Klotho was identified 15 years ago, its function remains incompletely understood. Recent studies of α-Klotho have unveiled numerous previously unknown molecular mechanisms of aging. Here, we present a review of the recent molecular, genetic, biochemical, and physiological properties that have been discovered for α-Klotho to date. Specifically, this review discusses the gene structure of α-Klotho and the factors that regulate α-Klotho gene transcription, the key sites in the regulation of α-Klotho enzyme activity, the α-Klotho signaling pathways, and the molecular mechanisms underlying α-Klotho function. Thus, this review will promote the current understanding of the molecular biology of α-Klotho, provide new insights into the function of α-Klotho, and offer future directions for α-Klotho research.
II. Klotho Gene
A. KL gene localization
The Klotho (KL) gene encodes the α-Klotho protein. Information about the KL gene and its protein is provided in Table 1. The KL gene was identified by Kuro-o et al (1) in 1997 through a spontaneous mutation of the α-Klotho promoter region of α-Klotho. The mouse KL gene locus is located on chromosome 5 (Figure 1) and flanked by PDS5B and STARD13. The locus of KL gene on chromosome 12 in rats and on chromosome 13 in humans are syntenic, as the neighboring genes are the same in both species as in the mouse (17).
Figure 1. Architecture of the Klotho gene and protein from the human, mouse, and rat.
A, The structure of the Klotho gene from the human, mouse, and rat. The upstream and downstream neighboring genes are highlighted in light brown. Introns and exons are highlighted in blue and green, respectively. B, Architecture of the human Klotho protein. Protein models were examined using the NCBI Conserved Domains and Protein Classification server.
Genes homologous to KL have been identified in D rerio and C elegans. In D rerio, the KL gene is located on chromosome 10, and the neighboring genes are Fryb and LOC100534680, which are homologous to the human and mouse PDS5B and STARD13 genes, respectively (18). Two prospective orthologs of α-Klotho, C50F7.10 and E02H9.5, have also been identified in C elegans (19). The two genes are located on different chromosomes and share 79% identity. It has been shown that disruption of the expression of one gene does not cause any obvious changes in phenotypes; however, a double-knockout model is currently unavailable in C elegans (19).
B. KL promoter characteristics
The predicted transcription factor binding sites of KL are shown in Figure 2. Several consensus sequences have been predicted to bind to different transcription factors. The human KL promoter region is an Sp1-rich region that cooperates with Oct-1 (20) and enhances the downstream KL gene expression, but it does not have a TATA box. The missing TATA box in the KL promoter is a feature that is similar to the kidney-specific cadherin (Ksp-cadherin) promoter (21). Ap-2 is able to bind to the enhancer region of simian virus 40 and stimulates RNA synthesis (22). PAX-4 is a major transcription factor in pancreatic cells and a crucial regulator in mammalian pancreas development (23). Mzf1 is a bifunctional regulator that represses transcription in nonhematopoietic cells and activates transcription in cells of hematopoietic origin (24, 25). E-box is a binding site for transcription factors in which a DNA sequence is recognized by proteins such as c-Myc, E47, and Myo-associated enzymes, which regulate the downstream gene transcription. Lyf-1, which is also known as Ikaros family zinc finger protein 1, is an essential transcription factor in the hematopoietic system, especially in lymphocyte development (26).
Figure 2. Structure of the Klotho promoter.
A comparison of the structure of the human (A) and mouse (B) Klotho promoter regions. The match program was used to analyze the transcription factor binding sites (underlined). The EMBOSS CpGPlot/CpGReport/Isochore program was used to screen the CpG islands (highlighted in blue). The transcription coding sequences are indicated in uppercase italic characters.
Turan and Ata (27) analyzed the human KL promoter region and reported that the 500-bp region located immediately upstream of the KL transcription initiation site is a key regulator of KL transcription. In HEK293 cells, the initial 300 bp of the promoter region inhibits KL transcription through an unknown mechanism, whereas the remaining 200-bp region enhances KL expression (27). E-box, Lyf-1, and Ap-2 are all located in the initial 300-bp region, which suggests that these transcription factors may inhibit KL transcription (Figure 2).
The mouse KL promoter region is also Sp1-rich and TATA-box-free. The mouse 300-bp region located immediately upstream of the start codon shares 67% identity with the same region in humans. Furthermore, the transcription factors Sp1 and PAX-4 are conserved in this region. Additional transcription factors in the mouse KL promoter region include GR, GATA, GKLF, AP-4, AREB4, and p300 consensus sites (Figure 2B). GR can cooperate with Sp1 and coregulates KL expression (28). GATA synergistically interacts with Sp1 (29). GKLF competes with Sp1 and interacts with the p300 transcription coactivators (30, 31). The expression of TGF-β, p53, and Sp1 is positively correlated with KL expression (27, 32). TNF-like weak inducer of apoptosis promotes RelA binding to the KL promoter, which may induce its deacetylation (33).
The KL promoter region is GC-rich (Figure 2) and sensitive to DNA methylation. Several research groups have reported that the methylation state of the promoter region is related to α-Klotho mRNA expression (34–37), suggesting that KL activity may be regulated by DNA methylation. The α-Klotho promoter region has a strong CpG-island methylation pattern in several cancers, including cervical, colorectal, gastric, and breast cancer, which suggests that α-Klotho may function as a tumor suppressor (38–43). One predicted CpG island (Figure 2, in blue) in the 500-bp human KL promoter region is 438 bp in size and has an observed-to-expected CpG ratio of 0.94. Two CpG island regions are predicted in mice. The CpG island that is farther from the start codon is 66 bp in size and has an observed-to-expected CpG ratio of 1.06. The CpG island closer to the start codon is 126 bp in size and has an observed-to-expected ratio of 1.08.
C. KL gene structure
There are five exons and four introns in the coding region of KL in humans, mice, and rats, which transcribe 3036, 3042, and 3042 nucleotide mRNAs, respectively (Figure 1). In addition, the mouse and human KL genes encode a short-form, secreted α-Klotho protein that is generated from translation of mRNA generated by alternative mRNA splicing (1, 17). Secreted α-Klotho is translated from 1650 and 1647 nucleotide mRNAs in humans and mice, respectively. Secreted α-Klotho has not been detected in rats.
D. Other KL gene properties
The KL gene has been reported to encode two other Klotho proteins, β-Klotho and Klotho-related protein (Klrp) (44, 45). Klrp is a transmembrane protein that binds to fibroblast growth factor receptor (FGFR) 1b, FGFR1c, and FGFR2c. However, the function of this complex is unknown (46, 47). β-Klotho, which is highly conserved and localized to the cell membrane (48), is expressed predominantly in the liver and white adipose tissue (48). In contrast, α-Klotho is expressed mainly in the distal tubule epithelial cells of the kidney. β-Klotho forms a complex with FGFR1c and FGFR4 (49–51). Fibroblast growth factor (FGF) 21 and FGF15/19 act through the β-Klotho–FGFR1c and β-Klotho–FGFR4 complexes, respectively, to activate downstream signaling (49–51).
The expression of FGF21 is regulated by a number of transcription factors, including peroxisome proliferator-activated receptor (PPAR)-α (52–55). Several reports indicated that the FGF21-FGFR1c-β-Klotho complex activates ERK1/2 phosphorylation and promotes the formation of the FGFR4-FGF19 complex to reduce bile acid synthesis (49, 50, 53, 56–58), but the underlying mechanism is unclear. β-Klotho knockout mice that were generated via the disruption of the exon 1 coding sequence (45) displayed a different phenotype than that of FGF21 knockout mice (59–61), which suggests that FGF21 may act independently from β-Klotho. On the contrary, studies in which β-Klotho knockout mice were generated via the disruption of β-Klotho exons 1–4 demonstrated that FGF21 activity may be dependent on β-Klotho in vivo (53). Similar results are observed when β-Klotho is knocked out in adipose tissue and the nervous system (62). These conflicting results suggest that the FGF21/β-Klotho pathway may be more complicated than the FGF23/α-Klotho pathway. FGF21 function mainly involves metabolic regulation, including glucose uptake (63) and therefore may be a therapeutic target of obesity and fatty liver disease (64).
The FGF15/19-FGFR4-β-Klotho complex suppresses the synthesis of Cyp7a1, which is a rate-limiting enzyme in bile acid synthesis in the liver (65). The consistent phenotypes of FGFR4, FGF15, and β-Klotho knockout mice suggest that these genes may share the same signal transduction pathway or are in some fashion at the molecular level (45, 65, 66).
In summary, β-Klotho is predominately expressed in liver and adipose tissue. The major function of β-Klotho involves metabolic regulation, glucose uptake, bile acid synthesis, and fatty acid metabolism, which are independent of α-Klotho. Thus far, no secreted form of β-Klotho has been discovered. Although β- and α-Klotho are generated from the same gene, they share distinct functions. α-Klotho is mainly expressed in kidney distal tubule cells and regulates phosphate (Pi) absorption and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] activity. α-Klotho has extensive function, which is partially due to the secreted form of Klotho that circulates in the blood. The secreted α-Klotho may have direct actions on tissues or cells that do not express Klotho (eg, vascular endothelial cells and smooth muscle cells).
III. α-Klotho Transcription and Post-translational Modification
A. Transcription
α-Klotho is expressed primarily in the kidneys and brain choroid plexus (4, 10). α-Klotho expression in the kidney is regulated by several physiological and pathological factors, including sustained circulatory and acute inflammatory stress, long-term hypertension, diabetes mellitus, and chronic renal failure (13, 67–71).
Several factors suppress KL gene transcription. Angiotensin II (AngII) decreases α-Klotho mRNA and protein expression levels (72). In renal tubular epithelial cells, AngII up-regulates TGF-β, p38, and p53 expression but down-regulates Sp1 expression (32). The Sp1 transcription factors are key positive regulators in the promoter region of KL (Figure 2), which suggests that the AngII-induced decrease in α-Klotho expression may be due to the suppression of KL gene transcription. Although this mechanism is not clearly defined, several reports indicate that KL expression is correlated with oxidative stress (73–75). Furthermore, the effect of AngII on KL transcription can be eliminated by iron chelation. Iron produces a hydroxyl radical, which produces reactive oxygen species (ROS), increases oxidative stress, and reduces α-Klotho expression.
Nuclear factor κ-light-chain-enhancer of activated B cells is another transcription factor that inhibits KL transcription. Nuclear factor κ-light-chain-enhancer of activated B cells is controlled by indoxyl sulfate and inflammatory cytokines such as TNF-like weak inducer of apoptosis and TNF-α (33, 38, 76, 77). The mRNA and protein expression level of α-Klotho also negatively correlates with levels of matrix metallopeptidase 9, tissue inhibitor of metallopeptidase 1, and plasminogen activator inhibitor-1. However, the mechanisms by which this occurs remain to be determined (78).
α-Klotho mRNA levels also are decreased in the distal convoluted tubules under several physiological and disease states, such as diabetic nephropathy and dehydration (79). Most factors that decrease α-Klotho mRNA expression are associated with changes in the aging process, which suggests that α-Klotho plays important roles in aging.
Factors that increase α-Klotho mRNA expression levels have also been reported. The KL promoter region includes a SNP site, G395A, which is located 395 bp immediately upstream from the start codon. The A variant of this SNP is associated with enhanced KL transcription and plays an important role in the early dysfunction of vascular access and uric acid in hemodialysis patients (80–83). It was reported that this SNP is associated with essential hypertension in subjects who are nonsmoking females > 60 years of age (8).
Vitamin D indirectly regulates α-Klotho expression by binding to vitamin D responsive elements, which are located 31–46 bp upstream of the Klotho start codon. Epidermal growth factor also regulates KL expression via early growth response protein 1, which binds to the KL promoter region 45 bp upstream of the start codon (84, 85). PPARγ increases α-Klotho expression in the mouse kidney (86, 87) via PPAR-responsive elements that are located in the 5′ untranslated region 3686–3698 bp upstream of the KL start codon, which is far from the KL coding region and suggests that regulation occurs through an indirect mechanism. In addition, erythropoietin, ras homolog gene family A, rapamycin, statins, fosinopril, and losartan are all reported to increase α-Klotho mRNA expression level, but the underlying mechanisms are still unclear (88–92).
Recently, some small molecules have been shown to enhance Klotho promoter activity and increase Klotho protein expression (93). 3,3′,5-Triiodo-L-thyronine, N-[2-(1-cyclohexen-1-yl)ethyl]-6,7,8,9-tetrahydropyrido[1,2-e]purin-4-amine, N-(2-chlorophenyl)-1H-indole-3-carboxamide, and 2-(1-propyl)amino-11-chlorothiazolo[5,4-a] acridine are either a hormone or chemical compounds that stimulate α-Klotho expression at both the mRNA and protein levels (93, 94). Troglitazone also increases α-Klotho mRNA levels, but only in the kidneys (95).
B. Post-translational modification
Full-length α-Klotho protein is located in the cell membrane. Many membrane-based proteins are modified by N- or O-linked glycosylation and then translocated to the cell membrane. There is no evidence, however, for glycosylation modification of the α-Klotho protein. A few sites for N-glycosylation have been predicted based on its amino acid (aa) sequence.
Another interesting post-translational modification is the cleavage of α-Klotho in a process named α-cut (96) by α-secretases A disintegrin and metalloproteinase domain-containing proteins 10 and 17 (ADAM10 and ADAM17, respectively) and β-secretase β-APP cleaving enzyme 1 (BACE1). The cleaved product is a soluble α-Klotho protein (∼130 kDa) that lacks both transmembrane and intracellular domains (Figure 1B). The remaining fragment, which remains embedded in the cell membrane, is cleaved by γ-secretase. Another mechanism named β-cut, which is promoted by insulin stimulation (4, 97), cleaves α-Klotho between the KL1 and KL2 domains and generates two fragments (∼65 kDa each).
The α-, β-, and γ-secretases also cleave the transmembrane region of amyloid precursor protein (APP). APP is the precursor of Aβ, which is a component of the amyloid plaques that are associated with Alzheimer's disease. α-Klotho expression is regulated by APP (98), which suggests that ADAMs may be involved in the aging process.
IV. α-Klotho Protein Characteristics
A. α-Klotho size, sequence, and activity
The N-terminal region of the α-Klotho precursor protein contains a signal peptide domain from aa 1 through 33 that is rich in hydrophobic aa and induces α-Klotho translocation from the cytosol to the membrane. Three types of α-Klotho proteins have been detected: the full-length transmembrane α-Klotho, soluble α-Klotho, and secreted α-Klotho. All three proteins have been detected in humans and mice, but only the transmembrane and soluble α-Klotho proteins have been detected in rats (4, 17).
The full-length α-Klotho protein is a transmembrane protein that contains two separate glycosyl hydrolase domains, KL1 and KL2. The activity and function of α-Klotho is associated with these two domains. A 20-aa single transmembrane domain is followed by a 9-aa intracellular domain (Figure 1B). As discussed above, the membrane-bound full-length α-Klotho protein can be cleaved by the membrane-anchored proteases ADAM10, ADAM17, and BACE1. The truncated α-Klotho protein, which is also known as the soluble α-Klotho protein, is released from the cell membrane and contains either KL1 only or both KL1 and KL2. The cleaving process can be inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor (97), which suggests that PI3K may be involved in the cleavage process. After entering the urine and blood, soluble α-Klotho functions as a hormone (2, 4, 96, 97, 99). The 549-aa secreted α-Klotho protein contains an N-terminal signal peptide that is followed by the KL1 domain only.
Sequence analysis shows that the KL1 and KL2 domains of the full-length α-Klotho protein are similar to the glycoside hydrolase family 1 (GH1). The GH1 β-glycosidase family comprises the enzymes that hydrolyze glycoside linkage and release small carbohydrates. Two Glu sites are important for their catalytic process; one Glu site acts as a nucleophile to attack the anomeric center and forms intermediate products, and the other Glu site acts as an acid/base that hydrolyzes the intermediate products to produce the final products (100). The active sites of the human and mouse α-Klotho proteins are different (Figure 3). The acid/base site of the KL1 domain is replaced by Asn in both humans and mice, but the nucleophile site of the KL2 domain is replaced by Ser in humans and Ala in mice.
Figure 3. Sequence alignment of selective Klotho sequences.
aa are colored according to their chemistry (blue = acidic; red = G or P; dark red = basic; green = hydrophobic), and conserved positions are highlighted when their aa occupants conform predominantly to a chemical type. The active site “E” is labeled with an asterisk (*). Positions of the starting aa in the total proteins are listed.
Klrp is highly homologous to α-Klotho and hydrolyzes glucosylceramide (101). In humans and mice, the N-terminal region of the full-length α-Klotho KL1 domain is highly conserved to the Klrp nucleophile site region, and the C-terminal region of the KL2 domain is highly conserved to the Klrp acid/base region. The protein alignment results are shown in Figure 3. Noguchi et al (102) mutated the acid/base site of the KL1 domain from Glu to Gln to reveal that the Klrp catalytic process includes two steps and involves the double-displacement mechanism to form covalent glycosyl-enzyme intermediate products, as is observed in α-Klotho.
The two active sites of α-Klotho are approximately 650 aa residues apart. In contrast, the two active sites in Klrp and other GH1 family members are approximately 200 aa apart. The protein sequence of α-Klotho is highly conserved among the GH1 family of proteins, which hydrolyze the glycosidic bond, but α-Klotho does not have significant hydrolytic activity. For example, Tohyama et al (103) evaluated the glycosidase activity of α-Klotho by applying artificial substrates and found that soluble α-Klotho has a lower glycosidase activity than either bovine liver β-glucuronidase or almond β-glucosidase. Together these results suggest that α-Klotho may belong to another glycoside hydrolase family.
Soluble α-Klotho can increase the abundance of both the transient receptor potential cation channel subfamily V member 5 (TRPV5) and renal outer medullary potassium channel (ROMK) 1 in the membrane. This function is mediated by the sialidase activity of α-Klotho, which hydrolyzes α 2, 6-linked sialic acids from the N-glycosylation sites of TRPV5 and ROMK1. These proteins are both important Ca2+ and K+ reabsorption channels in the kidney epithelial cell membrane. Hydroxylation prevents the internalization of TRPV5 and ROMK (104–106). This function can be suppressed by a mutation in the N-linked glycosylation site of TRPV5 (104).
Secreted α-Klotho is the major form of circulating Klotho. The secreted α-Klotho protein contains only a signal peptide in the KL1 domain, in which the acid/base site is mutated and the nucleophile site is conserved. Circulating Klotho may function as a hormone and regulate the functions of cells or tissues that do not express Klotho (eg, vascular endothelial cells and smooth muscle cells). This proposed function may partially explain why mutation of the Klotho gene causes extensive aging phenotypes despite being expressed in only a few tissues (eg, kidney and the brain choroid plexus). Further studies are needed to determine the nature of the generation and regulation of secreted Klotho and the mechanism of action of secreted Klotho, including the identification and characterization of the binding sites or receptors of Klotho.
It should be mentioned that circulating Klotho may include both soluble and secreted Klotho. Soluble Klotho is generated due to cleavage of the membrane form Klotho by α- and β-secretases (α-cut, ∼130 kDa) and by insulin stimulation (β-cut, ∼65 kDa). Soluble Klotho regulates TRPV5 and ROMK1 channels. In contrast, secreted Klotho is generated due to alternative RNA splicing (∼65 kDa). The short-form soluble Klotho and secreted Klotho both contain the KL1 domain and have approximately the same molecular weight (∼65 kDa). Although the function of soluble and secreted Klotho is not clear, both may act as hormones and regulate functions in tissues or cells that do not express Klotho. Future studies are warranted to explore the biochemical and physiological functions of circulating Klotho.
B. α-Klotho H/R mutation
A few functional point mutations of α-Klotho have been identified. A homozygous gene mutation, which mutates the coding region A to G at site 578 and changes the aa from His to Arg, was identified in a 13-year-old girl who had severe tumoral calcinosis with dural and carotid artery calcifications (107). This homologous mutation in the mouse KL gene decreases mouse α-Klotho expression, which can be recovered by exposure to low temperature. This mutation in the mouse suppresses the binding of membrane α-Klotho to FGF23 but does not affect α-Klotho binding to FGFR. The patient with this mutation exhibited some pathological phenomena, including hypercalcemia, hyperphosphatemia, and increased 1,25(OH)2D3 levels (107), which are symptoms similar to those of the α-Klotho mutant mouse.
C. α-Klotho KL-VS mutation
The KL-VS mutation is a well-studied polymorphism that refers to the human α-Klotho F352V and C370S mutations. The F352V mutation affects the distribution of secreted α-Klotho; this mutation diminishes the secretion of α-Klotho but increases the abundance of secreted α-Klotho on the cell membrane. The molecular weight of freely secreted α-Klotho is approximately 5 kDa larger than that of the membrane-bound secreted α-Klotho. This difference results from variable post-translational modification. The C370S mutation increases secreted α-Klotho but does not affect the membrane-bound level of α-Klotho. In the double-mutated KL-VS, the distribution of α-Klotho is balanced between the secreted and membrane-localized forms (108–110). Both of these mutations are located in exon 2 of the KL gene and are also associated with other nonsense mutations. The mouse allele of Phe is the same as in humans, but the allele site of Cys is replaced by Ser. This difference suggests that the F/V mutation plays a more important role than the C/S mutation in determining Klotho function. Furthermore, Zhou et al (111) reported that the KL-VS mutation leads to an altered homodimerization of α-Klotho and indirectly changes the association between α-Klotho and FGFR1c, which indicates that the KL-VS mutation is important for determining α-Klotho function in mineral metabolism.
The KL-VS mutation has been associated with several diseases in humans. For example, the KL-VS heterozygous mutation is associated with an increased risk of breast and ovarian cancer among BRCA1 (185deleteAG, 5382insertC) mutation carriers (112). Nzietchueng et al (113) reported that the KL-VS mutation also was significantly associated with lower systolic blood pressure and pulse pressure in a French population, which is in conflict with the findings of Arking et al (109). Further studies are required to determine the relationship between the KL-VS mutant and cardiovascular function and disease.
V. Transmembrane and Soluble α-Klotho Functions
A. Inhibition of the insulin/IGF-1 signaling pathway
Several studies have reported that disruption of α-Klotho expression decreases insulin production and increases insulin sensitivity (114–117). α-Klotho knockout mice also exhibit less energy storage and less energy expenditure compared with wild-type mice (118). Several studies have shown that α-Klotho suppresses the downstream signaling pathway of the insulin receptor substrate (IRS) and the IGF-1 receptor (IGF-1R) without directly binding to these receptors (2, 73, 119), but the mechanism by which Klotho regulates IRS and IGF-1R activity remains unclear.
One clue as to how Klotho may regulate IRS and IGF-1R activity involves the forkhead box proteins (FOXOs). Activated IRS leads to activation of the downstream PI3K/Akt signaling pathway and phosphorylation of FOXO1, FOXO3a, and FOXO4. The phosphorylated FOXOs remain in the cytoplasm rather than the nucleus, which results in a loss of its transcriptional activity (Figure 4a). FOXO1 indirectly enhances the rate of hepatic glucose production by increasing the transcription of glucose-6-phosphatase expression (120). One report suggests that FOXO1 negatively regulates adipogenesis through its binding to the promoter region of PPARγ, which is required to initiate adipogenesis in the nucleus (121). Other data, however, suggest that α-Klotho may promote adipogenesis through CCAAT/enhancer-binding proteins (C/EBPs) and PPARγ (122), which collectively maintain adipocyte gene expression to terminate the differentiation of preadipocytes (123). The expression of one of the C/EBPs, C/EBPβ, is a marker of adipogenic enhancers in preadipocytes and is regulated by cAMP through the protein cAMP-dependent protein kinase (PKA)-phosphorylated cAMP-responsive element-binding (124). The Janus kinase 2, signal transducer and activator of transcription 3, and Krüppel-like factor 4 signaling pathways also directly promote C/EBPβ expression in committed preadipocytes (125–130). α-Klotho may therefore indirectly regulate the insulin/IGF signaling pathway (4), although the exact mechanism remains to be elucidated.
Figure 4. Overview of the functions of Klotho.
a, Klotho inhibits the IGF signaling pathway. The downstream factors FOXO1, -3a, and -4 mediate the function of Klotho. b, Klotho suppresses the Wnt signaling pathway. c, Klotho regulates Trpv5 calcium channels. d, Klotho regulates PTH synthesis. e, The Klotho-FGF23 complex regulates Pi absorption, mineral metabolism, and vitamin D3 expression and activity.
In addition, recent studies indicate that C/EBPs regulate PPARγ expression by binding to the PPARγ promoter region (131–133). It has been reported that α-Klotho up-regulates C/EBPβ expression (115). Chihara et al (122) found that α-Klotho also induced C/EBPα- and PPARγ mRNA expression, which in turn promotes adipocyte differentiation. In a future study, it would be interesting to assess whether α-Klotho regulates PPARγ expression through C/EBP activation in adipocytes.
B. Suppression of WNT signaling
α-Klotho regulates WNT signaling (4). Liu et al (134) reported that α-Klotho binds to different types of Wnt ligands to suppress the downstream signaling transduction and that the α-Klotho knockout increases Wnt signaling in mice. The activated Wnt3 signaling pathway prolongs the cell cycle by arresting the cell cycle at the G2/M phase and up-regulating fibrogenic cytokines. In contrast, the α-Klotho-treated cell bypasses this phase and exhibits reduced fibrogenic cytokine production (135). Moreover, the recombinant α-Klotho protein has been shown to inhibit Wnt5A internalization and signaling in cells that overexpress Wnt5A (136). Klotho may therefore suppress Wnt signaling (Figure 4b).
Under hypoxic conditions, α-Klotho expression is inhibited and β-catenin expression is up-regulated (137). Zhou et al (111) reported that Klotho may be a natural antagonist of endogenous Wnt/β-catenin and that the loss of Klotho may contribute to kidney injury by releasing the inhibition of pathogenic Wnt/β-catenin signaling. The in vivo expression of Klotho decreases the activation of renal β-catenin and improves renal fibrosis in chronic kidney disease (111). Zhou et al (111) further explored the mechanism of inhibition by performing an in vitro immunoprecipitation assay that identified a potential association between α-Klotho and Wnt1, Wnt4, and Wnt7a as a complex. β-Catenin can be degraded by μ-calpain, a calcium-dependent protease. α-Klotho regulates intracellular Ca2+ (Figure 4c), which in turn increases μ-calpain activity leading to the degradation of β-catenin. Uchihashi et al (138) reported, however, that the in vivo reduction of Wnt activity in Klotho mutant mice did not rescue or delay aging. Thus, the in vivo regulation of Wnt/β-catenin by α-Klotho, including its role in aging, requires further validation.
Chen et al (139) reported that the injection of soluble α-Klotho into α-Klotho mutant mice also did not fully rescue the α-Klotho deficiency-related premature aging phenotypes. This result suggests that membrane α-Klotho is more important than soluble α-Klotho in mouse development. Mouse development is largely controlled by stem cell proliferation. Because the Wnt signaling pathway is known to regulate stem cell proliferation, it will be interesting to assess in future studies whether the Wnt signaling pathway mediates the role of α-Klotho in stem cell development.
C. Participation in Ca2+ homeostasis
Chang et al (104) reported that soluble α-Klotho, which is derived by cleaving the full-length α-Klotho protein from the cell membrane, increases the abundance of TRPV5 on the cell membrane. This effect can be abolished by a single glycosylation site mutation. This effect also cannot be repeated in α 2, 6-sialyltransferase expression–lacking cells, such as Chinese hamster ovary cells. Activity can be recovered, however, by the overexpression of the recombinant α 2, 6-sialyltransferase (105). In addition, galectin-1 binds to β-galactoside-binding proteins, which suggests that galectin-1 is involved in the modulation of cell–cell and cell–matrix interactions. When galectin-1 expression is suppressed or its activation is inhibited, the abundance of TRPV5 on the cell membrane is reduced. Galectin-1 can bind to N-Acetyllactosamine (LacNAc) or α 2, 3-sialylated LacNAc but not α 2, 6-sialylated LacNAc (140). Together these results suggest that α-Klotho may have the sialidase function to remove α 2, 6-sialylated LacNAc from the TRPV5 N-glycosylation branch to expose the galectin-1 binding site. The exposed galectin-1 makes it possible for α-Klotho to modify the process by which TRPV5 forms a stable polymer and prevents the TRPV5 endocytosis process. The same modification was also discovered by Cha et al (106) for ROMK, an ATP-dependent potassium transporter on the cell membrane. ROMK membrane accumulation is dependent on α-Klotho sialidase activity (106) (Figure 4c).
In HEK293 cells that are cotransfected with TRPV5 and α-Klotho, Ca2+ uptake is increased in proportion to α-Klotho expression (79). In a transgenic mouse model with targeted disruption of the α-Klotho gene in the renal distal tubule, a decrease in TRPV5 expression in the distal tubular cells was accompanied by a mild increase in urinary calcium excretion (141). Klotho may therefore regulate calcium metabolism via TRPV5. Nevertheless, no significant association between α-Klotho and TRPV5 expression has been found in the kidney.
Some reports indicate that α-Klotho binds to vascular endothelial growth factor receptor-2 and transient-receptor potential canonical Ca2+ channel 1 (TRPC-1) through its KL2 domain and regulates TRPC-1-mediated Ca2+ flux entry to maintain endothelial integrity (142). A recent study indicated that α-Klotho increases the plasma membrane retention of TRPV2, leading to enhanced glucose-induced insulin secretion in pancreatic β-cells (15). Although most data suggest that α-Klotho sialidase activity is related to the Ca2+ channel activity, it remains unclear how α-Klotho removes α 2, 6-sialylated LacNAc. It will be of interest in future studies to assess how soluble α-Klotho regulates multiple ion channels and how the ion flux contributes to the aging process.
Imura et al (143) reported that α-Klotho and Na+,K+-adenosine triphosphate (Na+,K+-ATPase) can form a complex, which further translocates to the plasma membrane under the increased concentration of extracellular free Ca2+. The complex of α-Klotho and Na+,K+-ATPase regulates the synthesis and release of PTH (Figure 4d), which plays an important role in FGF23-induced production of 1,25(OH)2D3. Nabeshima (144) reported that Ca2+ homeostasis is maintained by a multistep response system with response times ranging from seconds to days. Among these steps, α-Klotho expression responds to Ca2+ concentration through Na+,K+-ATPase on the order of seconds. This result suggests that α-Klotho is a fast regulator of Ca2+ absorption. Drüeke (145) presented a model to explain this complicated mechanism.
D. Participation in phosphate homeostasis
Phosphate (Pi) is essential to a variety of physiological processes and is regulated by 1,25(OH)2D3, PTH, and fibroblast growth factor 23 (FGF23) (146–149). Pi absorption in the intestines is mediated by the type 2 family of sodium-dependent Pi cotransporter b (NaPi-2b) and 1,25(OH)2D3, which constitute 30% of total Pi (147). Pi reabsorption in the kidney is regulated by type 1 (NaPi-1), type 2 (NaPi-2), and type 3 (PiT) sodium-dependent Pi cotransporters. The type 2 family includes three carriers (NaPi-2a, NaPi-2b, and NaPi-2c), and the type 3 family includes two carriers (PiT1 and PiT2) (150, 151).
Pi absorption increases the secretion of FGF23, a bone-derived hormone that was identified as a gene associated with autosomal dominant hypophosphatemia rickets (149, 152). In autosomal dominant hypophosphatemia rickets patients, FGF23 carries a gain-of-function point mutation that causes resistance to the degradation of FGF23, which leads to elevated blood FGF23 levels. FGF23 is a critical hormone in the regulation of the renal excretion of Pi (153–155). FGF23 is composed of N-terminal and C-terminal fragments (156). Secreted FGF23 has a low affinity to its receptors, an observation that had been difficult to interpret until the function of α-Klotho was found. Three different FGF23 receptors (FGFR1c, FGFR3c, and FGFR4) have been identified on the cell membrane. FGFR3 and -4 are involved in vitamin D metabolism, and FGFR1 is involved in Pi reabsorption (157, 158). Full-length α-Klotho binds to FGFRs to enhance the binding ability of FGF23 (157, 159). The N-terminal region of FGF23 interacts with FGFRs, and the C-terminal region of FGF23 interacts with α-Klotho (107, 160, 161). This binding activates a MAPK cascade, which inhibits Pi reabsorption in renal proximal tubule cells and suppresses the expression of NaPi-2a and NaPi-2c as well as the Cyp27b1 gene that encodes 1-α-hydroxylase, which hydrolyzes 25-dihydroxyvitamin D3 to generate 1,25(OH)2D3 (159, 162, 163). Several studies have reported the formation of the FGF23-Klotho complex on the cell surface and the subsequent activation of the downstream signaling pathways, including the phosphorylation of Erk1/2, p38, JNK, AKT, IκB, and GSK-3β (158, 159, 164).
FGF23 activates the Cyp24a1 gene that encodes 24-hydroxylase, which inactivates 1,25(OH)2D3 (158, 159, 164–171) (Figure 4e). In addition, FGF23 is also able to inhibit PTH mRNA expression and PTH secretion in the parathyroid glands (172). PTH is an activator of Cyp27b1 expression, which further activates 1,25(OH)2D3, leading to the absorption of dietary Ca2+ (173). PTH not only promotes the reabsorption of Ca2+ but also reduces the reabsorption of Pi. In addition, PTH increases FGF23 mRNA levels via the activation of PKA through the PTH receptor and β-catenin (174). The circulating 1,25(OH)2D3 activates FGF23 expression in the bone by binding to the vitamin D receptor and the retinoid X receptor.
It has been shown in different mouse models that α-Klotho is essential to the regulation of Pi absorption by FGF23 in the proximal tubules. α-Klotho knockout mice (kl−/−) exhibit a marked increase in the serum levels of Pi, Ca2+, and 1,25(OH)2D3 as well as extensive premature aging phenotypes (175). Interestingly, FGF23 knockout mice exhibit identical aging phenotypes and evaluated serum levels of Pi, Ca2+, and 1,25(OH)2D3 (170, 176–178). These data suggest that the role of FGF23 in the regulation of Pi metabolism may be regulated by α-Klotho. It has been reported that a Pi-deficient diet reduced the serum Pi level and extended the life span of both Klotho and FGF23 knockout mice, despite a further increase in Ca2+ and 1,25(OH)2D3 levels (176, 179). The premature aging phenotypes also can be reduced in both gene knockout mice by a 1,25(OH)2D3-deficient diet or a knockout of VDR or Cyp27b1 genes (175, 176, 180, 181). FGF23 and α-Klotho double-knockout mice showed high serum Pi levels and identical aging phenotypes to the FGF23 or α-Klotho single-knockout mice (182). However, exogenous FGF23 did not affect the high serum Pi levels in the double-knockout mouse. Hyp mice possess a mutation that inactivates the Pi-regulating gene and are associated with severe hypophosphatemia due to increased serum levels of FGF23 (183). To explore further the relationship between α-Klotho and FGF23, Nakatani et al (183) generated a hypophosphatemia and α-Klotho double-mutant mouse (HYP/Klotho−/−). Interestingly, hypophosphatemia was reversed to hyperphosphatemia in the double-mutant mouse despite the high serum level of FGF23 (183). This finding was further confirmed by Brownstein et al (184). The role of FGF23 in the regulation of Pi reabsorption is therefore dependent on α-Klotho.
The protein expression level of NaPi-2a is increased in the proximal tubular epithelial cells of Klotho-knockout and HYP/Klotho mutant mice (182, 183). An NaPi-2a and α-Klotho double-knockout prevented hyperphosphatemia (185). The double-knockout mice regained their reproductive ability and body weight and also showed reduced organ atrophy and suppressed ectopic calcifications, which prolonged survival (185). The disruption of NaPi-2a resulted in increased Pi excretion in the urine and decreased serum Pi levels (hypophosphatemia) but further increased the elevated serum levels of calcium and 1,25(OH)2D3 in α-Klotho knockout mice (186–189).
Hyperphosphatemia promotes mammalian aging (185). Hyperphosphatemia damages tissues and organs (4, 190–193) and contributes to chronic kidney disease (191). Overall, Klotho deficiency increases Pi reabsorption in the kidneys, which results in hyperphosphatemia. In addition, Klotho deficiency increases bone absorption and Pi flux into the blood, which contributes to hyperphosphatemia. The expression of α-Klotho is significantly reduced in patients with chronic kidney disease (13). These patients also share similar pathological features with α-Klotho knockout mice and exhibit high levels of FGF23 (192, 193).
There are two approaches that can rescue α-Klotho deficiency-induced premature aging phenotypes in mice: the knockout of NaPi-2a expression (185, 186) and a low-Pi diet (176). A low-Pi diet limits Pi uptake (176). NaPi-2a ablation disrupts Pi reabsorption from the kidneys (186), which leads to the attenuation of hyperphosphatemia. Pi also regulates the life span of Drosophila (194). The restriction of Pi absorption or the reduced cellular uptake of Pi can extend the life span of adult Drosophila (194). High Pi levels therefore appear to play an important role in the aging process across different species. Taken together, the current data support the concept that high serum levels of Pi are largely responsible for premature aging due to the inactivation of Klotho.
It should be noted, however, that a low-Pi diet that maintains normal levels of serum Pi does not rescue all aging phenotypes. For example, a low-Pi diet does not rescue stress-related heart damage in KL(−/−) mice (190). Systemic circulating soluble Klotho inhibits TRPC6 currents in cardiomyocytes by blocking the PI3K-dependent exocytosis of TRPC6 channels. This observation suggests that Klotho has direct effects on cardiomyocytes independent of hyperphosphatemia. Klotho may therefore have functions other than the regulation of Pi homeostasis. Further mechanistic studies are needed to better understand the role of Klotho in aging.
E. Suppression of oxidative stress
FOXO3a up-regulates the expression of manganese superoxide dismutase (MnSOD), an important enzyme for mitochondrial antioxidant defenses in mammalian cells (2, 119, 195). FOXO3a functions as a negative regulator of mitochondrial ROS generation (196). α-Klotho increases FOXO3a phosphorylation, which suggests that α-Klotho may suppress ROS-related oxidative stress. Transgenic mice that overexpress α-Klotho exhibit higher MnSOD expression and lower oxidative stress as evidenced by lower levels of urinary 8-hydroxy-2-deoxyguanosine, a marker of oxidative DNA damage (73, 119, 196). The overexpression of α-Klotho decreases H2O2-induced apoptosis, β-galactosidase activity, mitochondrial DNA fragmentation, superoxide anion generation, lipid peroxidation, and Bax protein expression (197–199), a function that was reported to be related to the apoptosis signal-regulating kinase 1 (200). Wang et al (199) reported that α-Klotho down-regulates the expression of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase 2, a catalytic subunit of NADPH oxidase that transfers electrons from NADPH to the outside of the membrane. This report further demonstrated that α-Klotho attenuates AngII-induced superoxide production, oxidative damage, and apoptosis through the cAMP/PKA pathway (199). α-Klotho deficiency increases endogenous ROS generation and accentuates oxidative stress (198). An in vivo Klotho gene delivery has been reported to decrease the up-regulation of NADPH oxidase 2 activity and superoxide production and prevent the progression of spontaneous hypertension (9). Although the mechanism by which α-Klotho extends the cellular life span is not fully understood, a recent study indicates that low levels of ROS-related stress responses are beneficial to the organism and can extend the life span of a cell (201). This result suggests that the antiaging function of α-Klotho may involve ROS and its downstream signaling pathways.
F. Other functions of Klotho
Depressed α-Klotho expression augments the expression of senescence-associated proteins, including β-galactosidase, p16, p21, and p53 (27, 76, 139, 202–204). Soluble α-Klotho plays a crucial role in the regulation of the cell cycle through the synthesis of p15 and p21 (139, 205). Soluble α-Klotho may be involved in apoptosis, cell cycle, and immune processes. Wolf and colleagues (206) report that both the full-length and secreted α-Klotho proteins may serve as tumor suppressors and inhibit tumor proliferation through the regulation of the IGF-1 signaling pathway. Soluble α-Klotho directly binds to type-II TGF-β receptors and prevents TGF-β1 binding to cell-surface receptors, thereby blocking TGF-β1 signaling (205). Reduced α-Klotho expression aggravates renal interstitial fibrosis. Secreted α-Klotho expression abolishes the fibrogenic effects of TGF-β1 (71, 111).
VI. Circulating α-Klotho Function
Three circulating forms of α-Klotho have been identified: the α-cut product (∼130 kDa), the β-cut product (∼65 kDa), and secreted α-Klotho (∼65 kDa) (4). The major circulating form of Klotho is short-form Klotho (∼65 kDa). Secreted α-Klotho is generated by the alternative mRNA splicing of α-Klotho (17). Although α-Klotho is expressed mainly in the distal tubule cells of the kidney and brain choroid plexus cells (1, 172), mutations to the α-Klotho gene cause extensive aging phenotypes in nearly all organs and tissues. This observation suggests that Klotho may have effects in cells or tissues that do not express Klotho. For example, circulating α-Klotho directly regulates the functions of endothelial cells and cardiac myocytes (4, 190), and therefore may function as a hormone. The function of secreted α-Klotho is less understood than that of the transmembrane α-Klotho protein. However, because the binding sites and receptors of secreted α-Klotho have not been identified, it will be imperative in future studies to determine the nature and sequence of α-Klotho binding sites in order to identify the extensive functions of circulating α-Klotho.
The protein sequence of the secreted α-Klotho protein is similar to that of the KL1 domain of α-Klotho, except for the C terminus. The aa 535–549 (SQLTKPISSLTKPYH) in the α-Klotho protein are replaced with the sequence DTTLSQFTDLNVYLW in secreted α-Klotho (17). It is difficult to differentiate secreted α-Klotho from other short forms of Klotho (eg, truncated KL1) due to the highly conserved sequences between different Klotho forms. A specific antibody against the variant peptide fragment may help resolve this problem.
High expression levels of secreted α-Klotho have been associated with ovarian tumors and are also positively correlated with the expression of IGF-1 and IGF binding protein-3 but not IGF-2 (207). Wolf and colleagues (206) reported, however, that secreted α-Klotho inhibited pancreatic cancer cell growth by interfering with the phosphorylation of the IGF signaling pathway. An in vivo experiment showed that secreted α-Klotho has greater inhibitory effects on tumor cell growth than full-length α-Klotho. Thus, the relationship between secreted α-Klotho and tumorigenesis is inconclusive and may vary with the tissue of origin.
Studies have reported that soluble α-Klotho regulates Pi metabolism by decreasing the activity and protein expression of NPT2a and NPT2b (208, 209). In addition, soluble α-Klotho up-regulates FGF23 mRNA and protein expression and enhances FGF23 binding to FGFR3 (210, 211). Smith et al found that the overexpression of cleaved α-Klotho increases FGF23 levels, which leads to hypophosphatemia, hypocalcemia, and bone fractures (211). FGF23 is a potent phosphaturic factor (153–155).
VII. Summary and Perspectives
Several functions of α-Klotho have been identified: 1) full-length α-Klotho serves as a coreceptor of FGF23 and enhances FGF23 signaling to maintain mineral metabolism; 2) full-length α-Klotho may suppress Wnt signal transduction; 3) soluble α-Klotho modifies TRPV5 and ROMK; 4) full-length α-Klotho inhibits the activation of the IGF/insulin signaling pathway; and 5) full-length α-Klotho increases resistance to oxidative stress. These known functions and related mechanisms are insufficient, however, to understand fully how α-Klotho slows the aging process and extends life span. Experimental research on α-Klotho has been slow due to: 1) the lack of efficient, specific inhibitors to block α-Klotho activity in vivo and in vitro; and 2) the lack of specific methods to distinguish the different forms of α-Klotho.
Klotho is an aging-suppressor gene. The full-length α-Klotho protein is expressed primarily in the kidneys and the brain choroid plexus, which does not explain the extensive role of Klotho in other organs and tissues. Circulating Klotho has direct effects on tissues and cells that do not express Klotho, which partially explains why a mutation to the Klotho gene causes such extensive aging phenotypes. Circulating Klotho may be generated by alternative RNA splicing (secreted Klotho) and/or via the proteolytic cleavage of the transmembrane form of Klotho (soluble Klotho) (4). Klotho may therefore function as a hormone. However, the binding sites or the receptors for Klotho remain unknown. It will therefore be important in future studies to identify and characterize Klotho receptors and investigate its downstream signaling.
Acknowledgments
This work was supported by National Institutes of Health Grants R01 HL118558, R01 DK093403, R01 AG049780, R01 HL105302, and R01 HL102074.
Disclosure Summary: The authors have no conflicts of interest to disclose.
Footnotes
- aa
- amino acid
- AngII
- angiotensin II
- APP
- amyloid precursor protein
- C/EBP
- CCAAT/enhancer-binding protein
- FGF
- fibroblast growth factor
- FGFR
- FGF receptor
- FOXO
- forkhead box protein
- GH1
- glycoside hydrolase family 1
- IGF-1R
- IGF-1 receptor
- IRS
- insulin receptor substrate
- Klrp
- Klotho-related protein
- LacNAc
- N-Acetyllactosamine
- MnSOD
- manganese superoxide dismutase
- Na+,K+-ATPase
- Na+,K+-adenosine triphosphate
- NaPi-2a
- sodium-dependent Pi cotransporter 2a
- 1,25(OH)2D3
- 1,25-dihydroxyvitamin D3
- Pi
- phosphate
- PI3K
- phosphoinositide 3-kinase
- PiT
- type 3 sodium-dependent Pi cotransporter
- PKA
- cAMP-dependent protein kinase
- PPAR
- peroxisome proliferator-activated receptor
- ROMK
- renal outer medullary potassium channel
- ROS
- reactive oxygen species
- SNP
- single-nucleotide polymorphism
- TRPC-1
- transient receptor potential canonical Ca2+ channel 1
- TRPV5
- transient receptor potential cation channel subfamily V member 5.
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