Klotho the Anti-Aging Molecule

Klotho: A Reciprocal ₪ Regulator of Aging & Disease

The research described below and on the next few pages, provides an in-depth exploration into the progress made in identifying molecules found in blood, having age-regression properties. When obtained from young animals and introduced into older organisms, they can produce profound health improvements, mitigate or even resolve age-related diseases, and effectively reverse biological aging. It is the intent of this introduction to guide the reader down a reductive path, arriving at what we now believe is a principal candidate for the rate limiting, molecular control of aging.

◉ Klotho plays a crucial role in multiple biological and immunological processes, and its strong correlation with aging, diseases of aging, and mortality, make it a compelling candidate fulfilling the requirements identified above as a key candidate for therapeutic intervention.

◉ High levels of Klotho extends health-span and lifespan. With age, our Klotho levels naturally decline, a trend that aligns closely with the onset of age-related diseases. (See the graphics* at right.)

◉ This striking Klotho/Age correlation is not mere coincidence; a growing body of scientific evidence robustly supports the idea that boosting Klotho levels is a strategic, effective intervention against aging. To underscore this, multiple companies are rushing to get a therapeutic to market that augments or up-regulates Klotho.

◉ The systemic plasma levels of Klotho, acting as a nexus between cellular regulation and systemic homeostasis, have been determined to have profound implications in the onset and progression of aging-related diseases, thereby establishing Klotho's role as an essential therapeutic target in the battle against the degenerative conditions of aging.(See graphic at right.)

◉ Klotho’s  appropriate signaling modulation on multiple crucial pathways, selectively and appropriately up or down regulates key aging molecular pathways and feedback loops, and its effects on critical biological processes such as oxidative stress, inflammation, platelet factor 4, and cellular senescence place Klotho in a dominant position in the control of aging.

◉  Klotho has been proposed as a biological marker of aging. The correlation between aging and Klotho is so strong that the two terms could be used interchangeably, i.e., your Klotho level is your age. 

[2015] Soluble aKlotho as a candidate for the biomarker of aging

Klotho declines with age in all mammalian organisms. Mice that were engineered to overexpress this gene lived 20-30% longer than normal mice, while Klotho-deficient mice showed a premature aging-like syndrome and had a significantly shortened lifespan. (See image on the right.)

Augmenting Klotho has definitively demonstrated dramatic health and age regressive benefits. This makes Klotho an exceptionally compelling target for anti-aging intervention, given its immediate targetability and the vast range of therapeutics, vitamins, and supplements proven to increase its levels. We cite here, over 100 available interventional opportunities that have demonstrated their ability to increase Klotho levels, all derived from published, peer reviewed research.  An optimal combination of active-agents that increase Klotho levels is still being determined. These pages are both a guide and a resource to anyone who would like to contribute to the goal of identifying an optimal set of Klotho increasing active-agents.  

• Mice given an abundance of the Klotho protein were healthier and lived longer. 

• Mice deprived of the protein had shorter lifespans.

[2022] Low Serum Klotho Associated With All-cause Mortality Among a Nationally Representative Sample of American Adults

For the last two decades it was well known that joining two similar animals of different ages (Heterochronic Parabiosis) imparted dramatic benefits to the old animal.  The quest to isolate the anti-aging molecules responsible for this benefit is detailed in this section.  Multiple research groups have narrowed this quest down to a single molecule: Klotho.

The journey to uncover the biological processes controlling aging has been a complex and winding path, sometimes resembling a random walk. The importances and relevance of the discovery of Klotho seems not to have been initially comprehended. In fairness to the authors of that 1997 study, effective age-regression by any means was completely contradictory to currently held scientific convention.

1863 • In the beginning ~

The first convincing evidence that any interventional strategy could positively impact the aging process comes from studies of Heterochronic parabiosis (HP). Heterochronic parabiosis is an experimental technique that involves the surgical attachment of two organisms, most commonly mice or rats of different ages, allowing them to share a common circulatory system. The technique has been employed since 1863 when Paul Bert described the technique in his doctoral theses. HP allows for the study of various physiological and pathological processes, such as organ preservation, cancer, immune responses and aging.  All published research titles, are active links to the full text when available. 

[1863] Theses - Animal Transplant, “De la Greffe Animal” P.Bert

Although thousands of studies incorporating HP have occurred since Paul Bert’s first describe the technique, the studies we cite below, lead us down a narrower and narrower pathway in an attempt to discover what the beneficial agent(s) are, within blood that controls aging. This guided scientific tour illuminates our current understanding of the biology and control mechanisms of aging.

1972 • Its in the blood ~

In 1972 a study was conducted to determine if the older mouse, when conjoined with a younger mouse would actually live longer. This resulted in the older mouse living for approximately 30 days longer than normal controls. This represents a significant lifespan extension for a mouse and equates to approximately 2 years in humans.

[1972] Mortality in a Synegenic Rat Parabiots of Different Chronological Age

2005 • Its in the blood ~

In a 2005 publication, Thomas Rando, Irene, and Michael Conboy from Stanford University, identified a phenomenon where old mice, when paired with younger ones, exhibited greatly improved muscle regeneration. This capability is generally only seen in younger mice. This pivotal finding immediately spurred an intensive search for the precise components within young blood, capable of resetting the biological age of the older animals used in these studies.

[2005] Rejuvenation of aged progenitorcells by exposure to a youngsystemic environment

2011 • Its in the blood ~

This HP study again conducted by Rando and Wass-Coray was focused on the negative impact that old blood has on the neurogenesis and cognitive function of the young mouse. This group demonstrated that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing a young mouse to an old systemic environment or to plasma from old mice decreased synaptic plasticity, and impaired contextual fear conditioning and spatial learning and memory.

[2011] The ageing systemic milieu negatively regulates neurogenesis and cognitive function; Saul A. Villeda, Thomas A. Rando & Tony Wyss-Coray 

2014 • Its in the plasma of the blood ~

Researchers at Stanford University, again led by Tony Wyss-Coray, demonstrated that infusions of plasma (the liquid part of blood without cells) from young mice could improve cognitive function in old mice. Our data indicated that exposure of aged mice to young blood late in life is capable of rejuvenating synaptic plasticity and improving cognitive function. The fellowing study is important because it begins the process of narrowing or focusing the locations of the candidates that are the primary agents in control of aging.

Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice

Harold Katcher & Elixir (E5)

2018 • What Specific Component(s) of Plasma Regulate Aging ~

With the insights provided by the 2005 publication by the Conboy’'s, and subsequent publications, Harold Katcher, Chief Scientific Officer of Yuvan Research, applied his intellect and insights into the root causes of aging, seeking to identify what the specific chronokines were within the plasma that imparted the restorative, youthful benefits.  In 2018 Harold first utilized a preliminary study as a probe to determine if modulating a specific and targeted set of molecular pathways would result in the same level of age-regression that HP or whole plasma accomplishes.

“Our first pass was to try a combination of known herbal supplements that are known to bind with the targets we’d identified. We gave them to rats, and at first nothing seemed to be happening. But after two months (about 4 years in human terms) the rats showed signs of rejuvenation. We were encouraged. Rather than continue with the herbs, though, we formulated the elixir, now E5 that we report on here. This is our first iteration, with dosage and timing determined theoretically, yet to be optimized in the lab.”

In results published by Dr. Katcher and Akshay Sanghavi in 2019, he reviewed the isolation of specific molecules contained within the plasma, capable of modulating the same molecular pathways identified utilizing the supplement formulation.  This information combined with Harold’s insights into the molecular biology of aging, enabled his to devise a process that allowed for the removal of a targeted sub-set of plasma fractions that would impact aging.   These fractions, are selectively isolated by molecular weight and size gradients. (If you know the molecular pathways, you know the signaling molecules that target it. Knowing the targets informs you of there molecular weight and size. This study was designed to replicated the HP and plasma studies described above, but with a targeted sub-set of plasma fractions harvested from young animals and injected into old animals.

The complete historical background of the benefits of age-regression observed in this preliminary study are outlined on this site and can be reviewed here. The results were both positive and dramatic enough (54% reduction in age as measured by epigenetic clocks) that many in the scientific community, like Steve Horvath and David Sinclair were at first skeptical of the results. David’s skepticism and eventual acceptance is detailed in this twitter thread. However, the specific active chronokines responsible for the anti-aging effects noted in their studies remain undisclosed by Katcher and his associate, Akshay Sanghavi.

[2019] Reversing age- dual species measurement of epigenetic age with a single clock

In a subsequent publication Dr. Katcher has identified Extracellular Vessels (EV) as the isolates obtained from the young pigs. The primary age-regressive signaling cargo of those exosomes is in all probability Klotho.  This is detailed below.

[2023] Reversal of Biological Age in Multiple Rat Organs by Young Porcine Plasma Fraction

By simplifying a very complex and technically demanding procedure, eliminating the need to surgically conjoin two animals, Harold and Akshay have moved this treatment from the lab, into the realm of practical commercialization.

There is a high probability that having a good understanding of the molecular targets impacted by the nutrational supplements utilized in their preliminary study, provides Harold and Akshay with a window of the plasma fractions that would prove to be the most beneficial to the success of future studies. This puts them in possession of a core inventory of both nutritional substances and plasma components that are effective to varying degrees at imparting age-regression benefits to the recipient. It should be noted here that biosimilar plant derived agents will never be as potent as the organismal derived agents, harvested from plasma, targeting the same molecular bio-identical targets/pathways. This observation does not preclude the ability of effectively modulating the molecular pathways responsible for aging with supplements, previously approved drugs, and vitamins, it just raises the bar to safely and effectively achieving that goal.

2018 • A Key Age Controlling Candidate Pops Up - in 1997 ~

Klotho is a key aging determinant as demonstrated by the linked studies referenced below. Heterochronic blood exchange (HBE) has demonstrated that circulating factors restore youthful features to aged tissues. However, the systemic mediators of those rejuvenating effects remain poorly defined. These authors of this study demonstrate that the beneficial effect of young blood on aged muscle regeneration was diminished when serum was depleted of extracellular vesicles (EVs). Whereas EVs from young animals rejuvenate aged cell bioenergetics and skeletal muscle regeneration.  Again, connecting the dots; the 2005 study by the Conboy’s demonstrating muscle rejuvenation in old mice after sharing a circulatory system was a pivotal article because it inspired intense interest in identifying exactly what the factors in blood/plasma were that conveyed this benefit. This study and the studies cited below, demonstrate that EVs play a key role in the rejuvenating effects of HBE and that Klotho transcripts within EVs phenocopy the effects of young serum on aged skeletal muscle. As the image on the right vividly demonstrates messenger RNA scripts coding for the production of Klotho decline with age. This mirrors the decline of soluble Klotho levels as we age.  

Image Source: Young blood" particles that help old mice fight aging identified

[2021] Regulation of aged skeletal muscle regeneration by circulating extracellular vesicles

Harolds and Akshay’s patent pointedly identify EV’s a one of the types of exasomes captured by their process.

The composition of claim 24 or pharmaceutical composition of claim 23, wherein the composition comprises extracellular vesicles, exosomes, exomeres, nonmembrane bound proteins, exogenous proteins and other molecules and molecular complexes. That all folks ;√) If it’s contained within the plasma, this sentence covers it.

EV’s contain a large number of signals that communicate disease state, age and immunological activation signals from the originating cell. It represents both a localized and systemic communication system.

[2019] Immune Cell-Derived Extracellular Vesicles – Functions and Therapeutic Applications

ESC-EVs have the therapeutic potential of ESCs and successfully avoid immunogenicity [31] and tumourigenesis [32], which can be used as an ideal cell-free alternative to ESCs in clinical treatment.

[2023] Embryonic stem cell-derived extracellular vesicles rejuvenate senescent cells and antagonize aging in mice

Multiple EV components have age regressive benefits. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) has demonstrated a NAD boosting potential. Using EVs to deliver eNAMPT to elderly mice has also been found to alleviate age-related tissue function, and to significantly extend lifespan.

[2019] Extracellular Vesicle-Contained eNAMPT Delays Aging and Extends Lifespan in Mice

It import to note here that the transmembrane form of Klotho would not be captured by the size/weigh gradient of Harold Katcher’s patent because they are bound within the cells bi-lipid membrane layer. Soluable Klotho proteins, messenger RNA and mRNA contained within an Extracellular Vesicle would be well within the size parameters listed in their claims.

2022 • EV’s are a Major Age Determining Factor ~ 

Several recent studies have identified expressed cellular information packets, also referred to as extracellular vessels (EV) and exasomes, as age determining circulating factors.  Our focus is obviously on Klotho as a key age determinant, but this article makes the point that isolation of EV’s from young animals contain a disproportionate spectrum of anti-aging signaling elements. EV’s are produced when an accumulation of specific molecules collect within and against the cells bi-lipid membrane walls. A physiological  event triggers this accumulation to bud away from the inner cell wall, producing and expelling a small packet of signaling molecules that will easily assimilate with other, similar, cell wall structures. When the two cell walls come into contact with each other, the fuse causing the signaling chronokinses contained within the EV to be injected directly into this new cell. EV’s can contain a wide variety of factors including cytokines, chemokine and mRNAs that can activate or inactive a vast number of targets. A good map of this process as it relates to aging can be viewed in the graphic on the right.

E5 is not just Klotho, but it is in all probability the most important single component of E5 or any other age impacting blood derived therapeutic.  As the complexity of the network effect of aging make evident, having the correct, adapted signaling milieu, made available by capturing the majority of the age-related signaling molecules as described in Harold and Akshay’s patent, will be a more powerful and effective therapeutic than any single agent approach addressing only one molecular target, even Klotho. Countering this interpretation is the fact that:

2023 • It Small Molecules in the Plasma ~

The most recent studies incorporating HP came to the conclusion that HP and blood exchange attest to the existence of signal molecules in blood plasma that regulate (or coordinate) aging.

[2023] Whole organism aging- Parabiosis, inflammaging, epigenetics, and peripheral and central aging clocks. The ARS of aging

[2023] Research Progress on the Anti-Aging Effect of Young Plasma and its Mechanism

This study demonstrated dramatic, broad, and enduring benefits, especially for the older mice, encompassing improved cardiac health, cognitive function, muscle repair and regeneration, and enhancements in organ functionality, specifically in the brain, liver, kidney, and pancreas.  More recently Epigenetic clocks have determined that the older mice did experience a regression of their biological age.

The Potential of Klotho as an Interventional Opportunity Preventing Diseases of Aging and Aging

Increasing Klotho levels holds the potential for dramatic improvements in immunity and disease prevention. For instance, an increased concentration of Klotho has shown potential in combating Alzheimer's disease, mitigating cognitive decline, and offering a protective shield for our memory centers. Likewise, in the realm of cardiovascular health, higher Klotho levels appear to counteract heart diseases, decreasing the likelihood of heart failure and atherosclerosis. It has also shown promising effects on kidney diseases, osteoporosis and even certain types of cancer. Thus, amplifying Klotho levels is like reorganizing and revitalizing our body's internal defense mechanisms to resist age-associated deterioration, thereby offering a captivating prospect in our quest for not just a lengthier, but a healthier and more vibrant lifespan.

Multiple studies have identified a large number of both molecular pathways and target molecules callable of modulating those pathways, all demonstrating anti-aging effects to varying degrees. One of the most effective targets; Klotho, is readably addressable, now, via nutraceutical interventions. Over the next few pages that inventory will be examined and discussed in detail as they relate to what we now believe is a key age controlling determinant: Klotho.