What is Klotho?
The klotho gene is composed of five exons and encodes a type 1 single-pass transmembrane glycoprotein (1014 and 1012 amino acids in mouse and human, respectively) that is located at the plasma membrane and Golgi apparatus. The intracellular domain is very short (~10 amino acids) without functional domains. The extracellular domain has two internal repeats, KL1 and KL2, which have amino-acid sequence homology to family 1 glycosidases that hydrolyze β-glycosidic linkage in saccharides, glycoproteins, and glycolipids. The linker region between two internal repeats contains four basic amino acids (Lys-Lys-Arg-Lys) that form a potential site for proteolytic cleavage.
Despite the sequence homology to glycosidase, glycosidase enzymatic activity is not detectable in recombinant Klotho protein probably because critical amino acid residues in putative active centers of the Klotho protein diverge from those of β-glycosidase enzymes. Indeed, Klotho exhibits weak β-glucuronidase activity in vitro and elicits biological effects through its β-glucuronidase and/or sialidase activity.
The extracellular domain of Klotho can be cleaved by membrane proteases such as ADAM10 and ADAM17 (ADAM metalloproteinase domain 10 and 17) and released into blood, urine, and cerebrospinal fluid. Cleaved Klotho functions as an endocrine, autocrine, and paracrine hormone on target cells. In addition, secreted Klotho is generated through alternative transcriptional termination of the klotho gene lacking exons 4 and 5 in mice. Secreted Klotho is detected in the blood, urine, and cerebrospinal fluid.
Klotho is expressed in multiple tissues and cell types and at particularly high levels in the kidney. Klotho is abundantly expressed in the distal convoluted tubule in the kidney and choroid plexus in the brain. It is also expressed in the renal proximal tubule], parathyroid gland and several sex organs including the ovary, testis and placenta. Recently, Klotho was found to be locally expressed in the adventitial area of the aorta, supporting the vascular protective effect of the Klotho protein. The list of tissue-specific expressions of Klotho is currently being updated.
Klotho has subsequently been characterized as one of a family of related proteins. These are all single-pass transmembrane proteins that include α-, β-, and ⋎- Klotho isoforms, the latter two discovered based on their homology with α-klotho.
α-Klotho comprises five exons and structurally its cognate protein is composed of a large extracellular domain followed by a transmembrane domain and a small domain of 11 residues comprising the intracellular C-terminus. The extracellular domain comprises two repeat sequences termed KL1 and Kl2 which are generated by full-length transcript splicing and can be cleaved by the metalloproteases ADAM-10 and ADAM-17. Cleavage of the extracellular domain results in a soluble form of Klotho being released. Soluble Klotho is the main functional form in the circulation and is detected in the blood, urine, and cerebrospinal fluid.
β-Klotho is mainly expressed in the liver, but is also found in the kidney, gut and spleen. It regulates the activity of members of the fibroblast growth factor (FGF) family, including FGF-21 and FGF-19.
⋎-Klotho is expressed in the skin and the kidney and has yet to be ascribed defined functions.
Mechanism of Action (Explain further)
See the function of Klotho protein in the image to the right.
The transmembrane Klotho forms a complex with FGF receptor (FGFR) and functions as a co-receptor for FGF23 and plays a crucial role in the regulation of phosphate and vitamin D metabolism in the kidney.
On the other hand, the transmembrane Klotho is clipped by membrane-anchored proteases ADAM10 and ADAM17 just above the plasma membrane. The entire extracellular domain of Klotho is then released into blood, urine, and cerebrospinal fluid.
The secreted Klotho protein has a putative sialidase activity that modifies glycans of calcium channel TRPV5 on the cell surface. A similar mechanism may explain the inhibitory effect of secreted Klotho on growth factors including insulin, IGF-1, and Wnt. The ability of secreted Klotho to inhibit IGF-1 signaling may contribute to the anti-oxidative stress and anti-cancer properties of Klotho.
What have Research Studies Shown?
Scientific research has revealed that Klotho has been associated with the following:• Reduce levels of oxidative stress
• Improve mitochondrial function
• Reduce renal fibrosis
• Reduce inflammatory burden
• Mitigate the effects of premature aging
• Enhance cognitive functions
• TFEB activator
• Regulates autophagy lysosomal pathway
• Clearance of Aβ and phospho Tau
Klotho in Research (Expanded)
Klotho is an anti-aging protein with pleiotropic actions that exerts organ protection. Several lines of evidence support the notion that Klotho functions as a human aging-suppression molecule. Polymorphisms of KLOTHO are correlated with life span, coronary artery disease, atherosclerosis, and osteoporosis in humans. Klotho is also associated with severe calcinosis and stroke. Klotho deficiency is involved in acute and chronic kidney diseases, cancers, and salt-sensitive hypertension. The serum level of Klotho decreases with aging in humans. However, the biological function of Klotho and the way in which Klotho deficiency contributes to age-related diseases remain elusive.
The klotho gene encodes a single-pass transmembrane protein that forms a complex with multiple fibroblast growth factor (FGF) receptors and functions as an obligatory co-receptor for FGF23, a bone-derived hormone that induces negative phosphate balance. Defects in either Klotho or Fgf23 gene expression cause not only phosphate retention but also a premature-aging syndrome in mice, unveiling a potential link between phosphate metabolism and aging.
In addition, the extracellular domain of Klotho protein is clipped on the cell surface and secreted into blood stream, potentially functioning as an endocrine factor. The secreted Klotho protein has a putative sialidase activity that modifies glycans on the cell surface, which may explain the ability of secreted Klotho protein to regulate activity of multiple ion channels and growth factors including insulin, IGF-1, and Wnt. Secreted Klotho protein also protects cells and tissues from oxidative stress through a mechanism yet to be identified. Thus, the transmembrane and secreted forms of Klotho protein have distinct functions, which may collectively affect aging processes in mammals.
Coronary Artery Disease
Researchers have identified a novel genetic risk factor for early-onset CAD. The KL-VS allele of the KLOTHO gene displays a strong gene-environment interaction, and the risk associated with this allele is modulated by modifiable risk factors. Importantly, ∼25% of individuals are carriers of this allele. These results suggest that genotyping for the KL-VS allele will identify individuals who are at higher relative odds of CAD and who could benefit from a genetically tailored therapeutic strategy.
Chronic Kidney Disease
Although the causes of CKD are multifactorial, klotho deficiency is significantly associated with the development and progression of CKD and extrarenal complications. Many clinical and animal studies have suggested that when the klotho-deficient state in CKD is rescued, the renal function, morphologic lesion, and complications of CKD are obviously improved. For example, the administration of soluble klotho protein significantly attenuated UUO-induced renal fibrosis and suppressed the expression of fibrosis markers and TGF-β1 target genes, such as Snail and Twist.
Furthermore, klotho connected intermedin 1-53 to the suppression of VC in CKD rats, and klotho supplementation suppressed the renin-angiotensin system to ameliorate Adriamycin nephropathy. In addition, klotho protein appeared to suppress the epithelial-mesenchymal transition by inhibiting TGF-β and Wnt signaling. Therefore, klotho deficiency may not only be a pathogenic intermediate in the acceleration of CKD progression but may also be a major contributor to chronic complications, such as CKD-MBD and cardiovascular diseases in CKD. Conceivably, any therapy that restores the klotho level by supplementation with exogenous klotho and/or the up regulation of endogenous klotho production might be a novel treatment strategy for CKD.
The tumor suppressive activity of Klotho was first identified in breast cancer in 2008. Recent investigations have implicated that Klotho is extensively downregulated in several solid tumors, including cervical cancer, pancreatic cancer, melanoma, and several digestive neoplasms. In these malignancies, Klotho was elucidated to be a modulator of several signaling pathways, including the FGF signaling, insulin-like growth factor-1 receptor (IGF-1R), and Wnt pathways, which are also involved in the pathogenesis of hematological malignancies.
Taken together, our findings identified that Klotho performs as a tumor suppressor and modulator of IGF-1R signaling in the DLBCL. Overexpression of Klotho may be a predictive marker for favorable outcome in DLBCL. Klotho reinforces the response of DLBCL cells to chemotherapeutic drugs. Being an endogenous circulating hormone, the secreted Klotho could function as an active form and inhibit the tumor growth effectively both in vitro and in vivo. This study illuminates Klotho as a potential target for future therapeutic strategies.
The first investigation of plasma Klotho in the aspect of CVD was performed in 2011 by Semba et al. The analysis included the common cardiovascular risk factors such as age, sex, total cholesterol, HDL cholesterol, systolic blood pressure, and diabetes. Interestingly, the risk of CVD in adults with higher plasma Klotho was lower. Similarly, there was an association between plasma Klotho and CVD in fifty healthy volunteers without any known risk factors for cardiovascular disorders.
Early predictors of atherosclerosis such as the thickness of carotid artery intima-media, flow-mediated dilation of the brachial artery, and the thickness of epicardial fat were explored. The results showed that low serum Klotho level was associated with larger thickness of epicardial fat and carotid artery intima-media and lower flow-mediated dilation of the brachial artery. Thus, lower levels of serum Klotho should be considered as an early predictor of atherosclerosis.
The scheme of an expected mechanism by which Klotho protein is involved in cardiovascular diseases.
(a) Local deficiency of vascular-derived Klotho leads to calcification. It is related to the FGFR/FGF23 resistance, which in turn inhibits the anti-calcific effect of FGF23. An attenuated expression of Klotho protein in vessel wall reduces production of NO and increases formation of ROS. Therefore, an imbalance of Klotho and FGF23 leads to oxidative stress and endothelial dysfunction.
(b) Depletion of Klotho can promote the prooxidative, proinflammatory, proapoptotic activity and damage of cardiomyocytes in the state of CVD risk. Therefore, cardiac dysfunction and cardiomyopathy may be observed.
(c) Klotho deficiency and KL gene polymorphisms are the risk factors for cardiovascular disease and correlate with the development of atherosclerosis, CAD, MI, or LVH.
(d) An occurrence of cardiac hypertrophy and remodeling in the state of Klotho deficiency is related to oxidative stress. It is caused by the activation of p38 and ERK1/2 signaling pathways, as well as by the overexpression of TRPC6 channels in heart. The treatment with exogenous Klotho may provide protection against the fibrotic alterations.
(e) Klotho contributes to alleviation of cardiac dysfunction and pathological changes in toxemic and ischemic heart. The treatment with Klotho mitigates an inflammation, ROS generation, apoptosis, mitochondrial dysfunction, fibrosis, and hypertrophy. Klotho may induce the restoration of cardiac function and thus could be explored as a therapeutic factor in myocardial injury. FGFR, fibroblast growth factor receptor; FGF23, fibroblast growth factor 23; NO, nitric oxide; ROS, reactive oxygen species; CAD, coronary artery disease; MI, myocardial infarction; LVH, left ventricular hypertrophy; ERK1/2, extracellular signal-regulated kinase 1/2; TRPC6, transient receptor potential canonical 6; , induction; , reduction.
Kim JH, Hwang KH, Park KS, Kong ID, Cha SK. Biological Role of Anti-aging Protein Klotho. J Lifestyle Med. 2015 Mar;5(1):1-6. doi: 10.15280/jlm.2015.5.1.1. Epub 2015 Mar 30. PMID: 26528423; PMCID: PMC4608225.
Dan E. Arking, Diane M. Becker, Lisa R. Yanek, Daniele Fallin, Daniel P. Judge, Taryn F. Moy, Lewis C. Becker, Harry C. Dietz,KLOTHO Allele Status and the Risk of Early-Onset Occult Coronary Artery Disease, The American Journal of Human Genetics, Volume 72, Issue 5, 2003, Pages 1154-1161, ISSN 0002-9297, https://doi.org/10.1086/375035.
Zhou, X., Fang, X., Jiang, Y. et al. Klotho, an anti-aging gene, acts as a tumor suppressor and inhibitor of IGF-1R signaling in diffuse large B cell lymphoma. J Hematol Oncol 10, 37 (2017). https://doi.org/10.1186/s13045-017-0391-5
Olejnik A, Franczak A, Krzywonos-Zawadzka A, Kałużna-Oleksy M, Bil-Lula I. The Biological Role of Klotho Protein in the Development of Cardiovascular Diseases. Biomed Res Int. 2018 Dec 24;2018:5171945. doi: 10.1155/2018/5171945. Erratum in: Biomed Res Int. 2020 Sep 29;2020:1463925. PMID: 30671457; PMCID: PMC6323445.