Klotho Peptide Research: Metabolic, Cardiac and Neuroprotective

Scientifically reviewed by
Dr. Ky H. Le, MD

The information presented in this article is for educational and research purposes only, intended for laboratory professionals, researchers and collaborators. This content does not constitute medical or clinical advice.

In 1997, Dr. Makoto Kuro-o was performing an experiment and made a serendipitous discovery that would change our understanding of aging at a molecular level.

He had been trying to generate transgenic mice with hypertension by inserting a certain piece of DNA into the genome. However, due to an error, the transgene inserted in the middle of an unknown gene, disrupting it.

These mice showed a phenotype of accelerated aging, developing multiple organ failures and dying at 2 months of age, rather than the usual 2.5 to 3 years.

After 4-5 years of research, Kuro-o and his team finally discovered the disrupted gene and published their discovery in the journal Nature^[1]. They named the gene after the Greek Fate Klotho, the spinner of the thread of human life.

While this was an extremely useful deficiency phenotype, it was only part of the story. They discovered later that mice that genetically overexpressed Klotho genes also showed a longer lifespan than usual by 20-30% and an increased resistance to oxidative stress^[2].

It was this double relationship that placed Klotho as a longevity-controlling protein with a wide range of protective effects.

Key Research Insights

• α-Klotho was accidentally discovered in 1997 when mice lacking it aged rapidly and died young
• The protein exists in three forms: membrane-bound, secreted, and soluble circulating versions
• Klotho protects multiple cellular systems by reducing inflammation, oxidative stress, and cellular damage
• Research shows it modulates critical pathways affecting metabolism, heart function, and cognitive function

The Klotho Protein Family

There are three proteins in the klotho family.

α-Klotho is predominantly expressed in the kidney, specifically in the distal convoluted tubule and connecting tubule, where it modulates mineral metabolism and also acts as a systemically circulating humoral factor^[3].

β-Klotho acts as an obligate co-receptor for the endocrine hormones FGF19 and FGF21. It is expressed in metabolically active tissues, such as the liver, adipose tissue and discrete areas of the central nervous system. α-klotho acts on phosphate and calcium homeostasis via FGF23, while β-klotho regulates bile acid metabolism (FGF19) and glucose and lipid metabolism (FGF21)^[4].

γ-Klotho, which is encoded by the LCTL (lactase-like) gene, is the least studied member of the klotho family. It is highly and selectively expressed in brown adipose tissue and the eye and has the capacity to act as an additional co-receptor for FGF19 in cell culture^[5].

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What is α-Klotho?

α-Klotho is a type I single-pass transmembrane protein found in various forms.

Dr. Carmela Abraham, Professor Emerita of Biochemistry at Boston University School of Medicine, characterizes the protein’s scope: “Klotho is a protein possessing multiple beneficial and unique pleotropic functions by protecting organs against inflammation, oxidative stress, tumor growth, while its absence leads to age-related diseases and early death.”

This protein has a large extracellular domain, a membrane-spanning segment, and a short intracellular domain. The extracellular domain has two internal repeat domains (KL1 and KL2) with structural homology to β-glucosidase enzymes^[6]. The protein, however, has no enzymatic activity and serves instead as a non-enzymatic scaffold.

Three forms of α-klotho have been identified that serve unique research roles:

• Full-length membrane-bound Klotho (mKl) serves as a co-receptor for fibroblast growth factor 23 (FGF23)
• Soluble Klotho (sKl) can be created by proteolytic cleavage by the ADAM10 and ADAM17 metalloproteases
• An alternatively spliced secreted form can also be produced and contributes to the circulating pool of the protein

α-Klotho protein structure (source publication).

Mineral Metabolism and Calcium-Phosphate Homeostasis Research

α-Klotho serves as an obligatory co-receptor for FGF23 in regulating phosphate and calcium homeostasis.

The membrane-bound form complexes with FGF receptors (primarily FGFR1c) to enable FGF23 signaling. This suppresses renal phosphate reabsorption and inhibits vitamin D biosynthesis. The FGF23-α-Klotho axis represents a bone-kidney endocrine network where FGF23, produced by osteoblasts and osteocytes, signals through α-Klotho-expressing renal tubular cells^[7].

Soluble α-klotho directly regulates the epithelial calcium channel TRPV5 in the kidney. This regulation enables calcium reabsorption in the distal tubule through two distinct mechanisms. α-klotho increases TRPV5 cell surface expression in an N-glycan-dependent manner, while sialidase-mediated stimulation operates through lipid raft-dependent pathways independent of N-glycosylation^[8].

Studies in mice with distal convolution-specific klotho deletion showed that approximately 80% of circulating soluble klotho originates from late distal convoluted tubule and connecting tubule cells. Loss of this source results in profound calciuria, reduced bone mineral density, and decreased TRPV5 expression.

Cardiovascular and Vascular Function Mechanisms

Cardiovascular protective mechanisms of α-Klotho are mediated by its antioxidant and anti-inflammatory properties, as well as its ability to regulate vascular smooth muscle cells phenotype.

Within endothelial cells, klotho protein enhances cell viability and upregulates the expression of antioxidantenzymes such as superoxide dismutase, catalase, and heme oxygenase-1 (HO-1). In addition, it directly scavenges reactive oxygen species. Klotho protein activates the PI3K/AKT signaling pathway, which in turn upregulates the expression of Nrf2/HO-1^[9].

In the case of vascular calcification, the absence of klotho promotes pathological calcification of the vascular wall. Restoration of klotho expression can attenuate calcification through the activation of autophagy. Anti-calcification effects are also explained by inhibition of Wnt/β-catenin signaling pathway in vascular smooth muscle cells and thereby preventing the osteogenic transition of vascular smooth muscle cells^[10].

In the case of cardiac fibrosis and hypertrophy, klotho has been shown to interfere with transforming growth factor-β (TGF-β) signaling pathways. Klotho inhibits TGF-β-induced fibrotic responses and downregulates the expression of fibrotic markers, such as α-smooth muscle actin, collagen, and fibronectin, in cardiac tissue.

Cognitive Function and Neuroprotection Research

α-Klotho has neuroprotective and cognition-enhancing effects via several mechanisms.

Serum and cerebrospinal fluid concentrations of α-klotho are positively associated with scores on standardized cognitive tests. They are also predictive of cognitive status independent of age or apoE4 genotype. α-Klotho may prevent cognitive decline by preserving optimal synaptic function, inducing antioxidant defense, diminishing inflammation, promoting autophagy, and improving clearance of amyloid-β^[11].

In hippocampal neurons, klotho augments the expression of the GluN2B subunit of NMDA receptors in postsynaptic densities. This leads to an increase in the strength of NMDA receptor-dependent long-term potentiation, which is required for learning and memory acquisition^[12].

Neuroprotective effects against glutamate-induced excitotoxicity and amyloid-β-induced oxidative stress are also achieved through the regulation of redox system members, specifically peroxiredoxin-2 (Prx-2).

KL1 domain of klotho mediates cognition improvement in an activity-mimetic fashion, by reproducing the metabolic state induced by cognitive stimulation in hippocampus, generating the same metabolic changes that result in improved cognition in both young and aging subjects.

Insulin and IGF-1 Signaling Modulation

α-Klotho is a negative modulator of insulin and insulin-like growth factor-1 (IGF-1) signaling.

Soluble klotho inhibits the PI3K/AKT/mTORC1 signaling pathway by directly binding type 1 IGF receptor (IGF1R). The consequent effects of this inhibition are increased peroxisome proliferator-activated receptor α (PPARα) expression and maintenance of hepatic glucose and lipid homeostasis. The protein also modulates insulin secretion and has been implicated in inducing pancreatic β-cell health and stimulating lipid oxidation in the liver and adipose tissue^[13].

The inhibitory effect of klotho on IGF-1 signaling is involved in the regulation of longevity. Decreased activity of the insulin/IGF-1 signaling pathway is associated with extended lifespan in a number of organisms. The regulatory mechanism is thought to involve the modulation of downstream transcription factors such as FOXO and the activation of stress resistance pathways^[14].

Klotho is an inducer or inhibitor of autophagy depending on the physiological or pathophysiological context and acts on this process through the IGF-1/PI3K/Akt/mTOR signaling pathway.

Wnt/β-Catenin Signaling Inhibition

α-Klotho is an endogenous inhibitor of Wnt/β-catenin signaling, a pathway that regulates cell proliferation, differentiation, and tissue homeostasis.

Klotho overexpression negatively regulates Wnt/β-catenin signaling by promoting β-catenin degradation and decreasing nuclear translocation. This leads to the suppression of Wnt target gene transcription through proteasome-dependent mechanisms downstream of Dishevelled (DVL)^[15].

The ability of klotho to suppress Wnt/β-catenin signaling has implications for the inhibition of fibrosis in multiple tissues. In liver cancer cells, the klotho-mediated suppression of Wnt/β-catenin signaling induces apoptosis and decreases proliferation^[16].

Similar mechanisms are observed in vascular smooth muscle cells, where klotho prevents calcification by inhibiting the Wnt pathway.

Inflammatory Response Regulation

α-Klotho exhibits anti-inflammatory properties through modulation of multiple inflammatory pathways and cytokine production.

The protein suppresses pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) through interference with nuclear factor-κB (NF-κB) signaling. Studies show that inflammatory cytokines, conversely, downregulate klotho expression through NFκB-dependent mechanisms, creating a reciprocal regulatory relationship^[17].

In glial cells exposed to lipopolysaccharide-induced inflammation, α-klotho pretreatment decreases TNF-α and IL-6 production, reverts NF-κB activation, and reduces neuronal death induced by inflammatory mediators. The protein also modulates immune cell phenotype and function, including effects on monocytes, macrophages, T cells, and B cells^[18].

Soluble klotho serves as an inverse biomarker of systemic inflammatory states. Serum levels inversely correlate with markers of inflammation. The protein’s anti-inflammatory effects contribute to its protective functions in chronic kidney disease, cardiovascular disease, and neurodegenerative conditions.

Mitochondrial Function and Oxidative Stress

α-Klotho has multiple mechanisms by which it confers oxidative stress resistance and mitochondrial function.

The protein upregulates manganese-containing superoxide dismutase (Mn-SOD) and the transcription factors FoxO and Nrf2 to activate antioxidant defense systems. Klotho affects regulators of mitochondrial function such as mitochondrial uncoupling protein 1 (UCP1), B-cell lymphoma-2 (BCL-2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), transcription factor EB (TFEB), and peroxisome proliferator-activated receptor gamma (PPAR-gamma)^[19].

Age-dependent loss of α-klotho expression in muscle progenitor cells drives mitochondrial DNA damage and a reduction in cellular bioenergetics. Restoring klotho expression to youthful levels ameliorates mitochondrial dysfunction, protects mitochondrial DNA integrity and enhances the functional regeneration of aged muscle tissue. The protein induces mitochondrial biogenesis by activating the AMPK/PGC-1α pathway^[20].

Tumor Suppressor Functions

α-Klotho functions as a tumor suppressor across multiple cancer types, including liver, gastric, lung, colorectal, melanoma, and other malignancies.

The protein inhibits cancer cell proliferation, induces apoptosis, and suppresses tumor progression through modulation of key oncogenic signaling pathways. Klotho expression is frequently downregulated in cancer tissues through promoter hypermethylation, and reduced expression correlates with poor prognosis in many cancer types^[16].

The tumor suppressive mechanisms involve inhibition of the Wnt/β-catenin pathway, which reduces expression of proliferative genes including c-Myc and Cyclin D1. Klotho also suppresses the IGF-1/insulin signaling axis by reducing phosphorylation of IGF-1 receptor, insulin receptor substrate-1 (IRS-1), PI3K, Akt, and mTOR^[21].

In lung cancer cells, klotho overexpression promotes apoptosis through upregulation of pro-apoptotic genes including Bax while downregulating anti-apoptotic Bcl-2. The protein also sensitizes cancer cells to chemotherapy, particularly cisplatin-based treatments, by attenuating drug resistance through PI3K/Akt pathway inhibition^[22].

Potential In Vitro Research Applications

Research Application	Mechanism of Interest	Relevant Pathway
Vascular Calcification Models	Investigation of calcium-phosphate deposition in vascular smooth muscle cells and protective autophagy mechanisms	Wnt/β-catenin inhibition, FGF23 signaling
Neurodegenerative Disease Models	Study of NMDA receptor modulation, amyloid-β clearance, and synaptic plasticity in hippocampal neurons	NMDA receptor signaling, antioxidant pathways
Cancer Cell Proliferation Studies	Analysis of tumor suppressor mechanisms, apoptosis induction, and chemotherapy sensitization in various cancer cell lines	IGF-1/PI3K/Akt pathway, Wnt/β-catenin suppression
Metabolic Dysfunction Models	Examination of insulin sensitivity, hepatic glucose metabolism, and lipid oxidation in hepatocytes and adipocytes	IGF-1R interaction, PPARα regulation
Cardiac Fibrosis Models	Investigation of TGF-β pathway inhibition and fibrotic marker expression in cardiomyocytes	TGF-β signaling, inflammatory cytokine modulation

Research Grade α-Klotho Peptide at BioLongevity Labs

BioLongevity Labs provides research-grade α-klotho protein for in vitro laboratory studies, manufactured under USA GMP standards with triple third-party testing.

Each batch includes comprehensive analytical documentation with Certificates of Analysis from three independent certified laboratories. HPLC purity verification confirms >99% purity, while LC-MS provides molecular confirmation of protein identity.

All products are strictly for research use only and not for human consumption. Same-day shipping is available for orders placed before 12pm PT, with free shipping on orders over $400.

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References

1. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Springer Science and Business Media LLC; 1997. https://doi.org/10.1038/36285
2. Cheikhi A, Barchowsky A, Sahu A, Shinde SN, Pius A, Clemens ZJ, et al. Klotho: An Elephant in Aging Research. Oxford University Press (OUP); 2019. https://doi.org/10.1093/gerona/glz061
3. Morevati M, Mace ML, Egstrand S, Nordholm A, Doganli C, Strand J, et al. Extrarenal expression of α-klotho, the kidney related longevity gene, in Heterocephalus glaber, the long living Naked Mole Rat. Springer Science and Business Media LLC; 2021. https://doi.org/10.1038/s41598-021-94972-1
4. Aaldijk AS, Verzijl CRC, Jonker JW, Struik D. Biological and pharmacological functions of the FGF19- and FGF21-coreceptor beta klotho. Frontiers Media SA; 2023. https://doi.org/10.3389/fendo.2023.1150222
5. Kim JH, Hwang KH, Park KS, Kong ID, Cha SK. Biological Role of Anti-aging Protein Klotho. Institute of Lifestyle Medicine; 2015. https://doi.org/10.15280/jlm.2015.5.1.1
6. Saar-Kovrov V, Donners MMPC, van der Vorst EPC. Shedding of Klotho: Functional Implications in Chronic Kidney Disease and Associated Vascular Disease. Frontiers Media SA; 2021. https://doi.org/10.3389/fcvm.2020.617842
7. Sun F, Liang P, Wang B, Liu W. The fibroblast growth factor–Klotho axis at molecular level. Walter de Gruyter GmbH; 2023. https://doi.org/10.1515/biol-2022-0655
8. Nabeshima Y. Discovery of α-Klotho unveiled new insights into calcium and phosphate homeostasis. Proceedings of the Japan Academy Series B, Physical and biological sciences 2009;85:125–141.
9. Cui W, Leng B, Wang G. Klotho protein inhibits H2O2-induced oxidative injury in endothelial cells via regulation of PI3K/AKT/Nrf2/HO-1 pathways. Canadian Science Publishing; 2019. https://doi.org/10.1139/cjpp-2018-0277
10. Li L, Liu W, Mao Q, Zhou D, Ai K, Zheng W, et al. Klotho Ameliorates Vascular Calcification via Promoting Autophagy. Wiley; 2022. https://doi.org/10.1155/2022/7192507
11. Hanson K, Fisher K, Hooper NM. Exploiting the neuroprotective effects of α-klotho to tackle ageing- and neurodegeneration-related cognitive dysfunction. Portland Press Ltd.; 2021. https://doi.org/10.1042/ns20200101
12. Gupta S, Moreno AJ, Wang D, Leon J, Chen C, Hahn O, et al. KL1 Domain of Longevity Factor Klotho Mimics the Metabolome of Cognitive Stimulation and Enhances Cognition in Young and Aging Mice. Society for Neuroscience; 2022. https://doi.org/10.1523/jneurosci.2458-21.2022
13. Landry T, Shookster D, Huang H. Circulating α-klotho regulates metabolism via distinct central and peripheral mechanisms. Elsevier BV; 2021. https://doi.org/10.1016/j.metabol.2021.154819
14. Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, et al. Suppression of Aging in Mice by the Hormone Klotho. American Association for the Advancement of Science (AAAS); 2005. https://doi.org/10.1126/science.1112766
15. Xu Y, Sun Z. Molecular Basis of Klotho: From Gene to Function in Aging. The Endocrine Society; 2015. https://doi.org/10.1210/er.2013-1079
16. Sun H, Gao Y, Lu K, Zhao G, Li X, Li Z, et al. Overexpression of Klotho suppresses liver cancer progression and induces cell apoptosis by negatively regulating wnt/β-catenin signaling pathway. Springer Science and Business Media LLC; 2015. https://doi.org/10.1186/s12957-015-0717-0
17. Moreno JA, Izquierdo MC, Sanchez-Niño MD, Suárez-Alvarez B, Lopez-Larrea C, Jakubowski A, et al. The Inflammatory Cytokines TWEAK and TNFα Reduce Renal Klotho Expression through NFκB. Ovid Technologies (Wolters Kluwer Health); 2011. https://doi.org/10.1681/asn.2010101073
18. Nakao VW, Mazucanti CHY, de Sá Lima L, de Mello PS, de Souza Port’s NM, Kinoshita PF, et al. Neuroprotective action of α-Klotho against LPS-activated glia conditioned medium in primary neuronal culture. Springer Science and Business Media LLC; 2022. https://doi.org/10.1038/s41598-022-21132-4
19. Donate-Correa J, Martín-Carro B, Cannata-Andía JB, Mora-Fernández C, Navarro-González JF. Klotho, Oxidative Stress, and Mitochondrial Damage in Kidney Disease. MDPI AG; 2023. https://doi.org/10.3390/antiox12020239
20. Sahu A, Mamiya H, Shinde SN, Cheikhi A, Winter LL, Vo NV, et al. Age-related declines in α-Klotho drive progenitor cell mitochondrial dysfunction and impaired muscle regeneration. Springer Science and Business Media LLC; 2018. https://doi.org/10.1038/s41467-018-07253-3
21. Xie B, Zhou J, Shu G, Liu D cai, Zhou J, Chen J, et al. Restoration of klotho gene expression induces apoptosis and autophagy in gastric cancer cells: tumor suppressive role of klotho in gastric cancer. Springer Science and Business Media LLC; 2013. https://doi.org/10.1186/1475-2867-13-18
22. Chen B, Wang X, Zhao W, Wu J. Klotho inhibits growth and promotes apoptosis in human lung cancer cell line A549. Springer Science and Business Media LLC; 2010. https://doi.org/10.1186/1756-9966-29-99