Senolytics Restore α-Klotho in Mice and Humans

New senolytics data was released from Dr. James L. Kirkland’s Mayo Clinic lab and published in The Lancet [1].

Prior studies have shown that α-Klotho protein decreases with age in mice and humans [2,3]. It has also been demonstrated that mice that lack α-Klotho have shorter lifespans, cognitive impairment, sarcopenia, vascular dysfunction, osteopenia, cardiac hypertrophy and fibrosis, and physical dysfunction [2-9]. α-Klotho overexpression in mice is linked to increased lifespan, cognition, and skeletal muscle regeneration. These mice also had decreased diabetes-related inflammation and delayed age-related vascular decline [10-12].

We have previously discussed how low α-Klotho levels are associated with all-cause mortality and dementia. This study sought to determine if there is a causal link between α-Klotho and cellular senescence by determining the effect of senolytics on urinary α-Klotho levels in naturally aged, diet-induced obese, and senescent cell-transplanted mice. For the human portion of the study, urine from patients with idiopathic pulmonary fibrosis (IPF) were assayed for α-Klotho from a prior study [13].

α-Klotho is decreased by senescent cells via paracrine mechanisms

In three cell lines, the researchers examined both non-senescent and senescent cells. Treating the cell lines with activin A or Interleukin-1a, two notable components of the SASP, caused α-Klotho expression to decrease. To further determine causality, they transplanted a small number of senescent cells into the adipocyte progenitor cells into young mice, which decreased α-Klotho in the urine, cerebellum, and choroid plexus compared to controls.

Removal of a senescence marker in senescent cells increased α-Klotho

In old and young transgenic mice that do not strongly express the senescence marker p16Ink4a, kidney, brain, and urine α-Klotho was still lower in the older mice than the young. Furthermore, α-Klotho was increased when mice were treated with a compound that decreases p16Ink4a.

The SASP factors activin A and Interleukin-1a were elevated in the kidneys and brains of the old mice. Decreasing p16Ink4a caused their activitin A and Interleukin-1a to also be decreased, supporting the hypothesis that these two SASP factors contribute to age-related decline.

In vivo senolytic treatment increases α-Klotho levels

What Are Senolytics? Senotherapeutics for Senescent Cells

Senolytics work by targeting one of several pro-survival pathways that senescent cells use to evade apoptosis and cling onto life. One of the challenges in dealing with senescent cells is that there are a number of different populations of these cells in our tissues and organs, each using different pro-survival pathways. It could be that multiple senolytic drugs need to be used to remove senescent cells using different pathways to survive.
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When mice were treated with dasatinib plus quercetin (D+Q) along with fisetin, α-Klotho levels were increased in the urine. Young wild-type mice with diet-induced obesity, which have more senescent cells than lean mice, had increased α-Klotho in urine with D+Q treatment. In young mice with transplanted senescent cells, D+Q and fisetin also increased urinary α-Klotho.

In the older mice, D+Q increased α-Klotho in the cerebellum and choroid plexus parts of the brain. The mRNA of α-Klotho was also increased in the the brains of the older mice and the obese mice. Additionally, α-Klotho levels were inversely related to adipose senescent cells in the obese mice.

In human cells and young transgenic mice, α-Klotho was not transcriptionally upregulated

Neither D+Q nor fisetin increased α-Klotho in non-senescent human preadipocytes (fat cells) or astrocytes (brain cells). In young female transgenic mice, decreasing p16Ink4a levels or administering D+Q did not increase α-Klotho in the kidneys or urine.

“Thus, senescent cell targeting strategies do not appear to increase α-Klotho when senescent cell burden is low, consistent with increases in α-Klotho being due to removal of senescent cells, rather than other mechanisms.”

Humans treated with senolytics have increased α-Klotho levels

The researchers had previously shown in IPF patients that nine doses of D+Q over three weeks led to improved gait distance and speed five days after the last dose of senolytics. Additional parameters show improved short performance scores and the ability to rise out of a chair. Urinary α-Klotho was increased after D+Q treatment.

Conclusion

The authors conclude their study by describing its implications:

“Our study also opens a novel, translationally-feasible avenue for developing orally-active small molecules to increase α-Klotho, which may also be a useful biomarker for senescent cell burden or senolytic drug activity in clinical trials.”

Six of this study’s authors on this study have a financial interest related to research and patents on senolytic drugs that are held by Mayo Clinic. However, this is a well-designed study with a wealth of data, and it may help to bring senolytics to the bedside as therapies against age-related diseases.

Literature

[1] Zhu, Y., Prata, L., Gerdes, E., Netto, J., Pirtskhalava, T., Giorgadze, N., Tripathi, U., Inman, C. L., Johnson, K. O., Xue, A., Palmer, A. K., Chen, T., Schaefer, K., Justice, J. N., Nambiar, A. M., Musi, N., Kritchevsky, S. B., Chen, J., Khosla, S., Jurk, D., … Kirkland, J. L. (2022). Orally-active, clinically-translatable senolytics restore α-Klotho in mice and humans. EBioMedicine, 77, 103912. https://doi.org/10.1016/j.ebiom.2022.103912

[2] Maique, J., Flores, B., Shi, M., Shepard, S., Zhou, Z., Yan, S., Moe, O. W., & Hu, M. C. (2020). High Phosphate Induces and Klotho Attenuates Kidney Epithelial Senescence and Fibrosis. Frontiers in pharmacology, 11, 1273. https://doi.org/10.3389/fphar.2020.01273

[3] Faul, C., Amaral, A. P., Oskouei, B., Hu, M. C., Sloan, A., Isakova, T., Gutiérrez, O. M., Aguillon-Prada, R., Lincoln, J., Hare, J. M., Mundel, P., Morales, A., Scialla, J., Fischer, M., Soliman, E. Z., Chen, J., Go, A. S., Rosas, S. E., Nessel, L., Townsend, R. R., … Wolf, M. (2011). FGF23 induces left ventricular hypertrophy. The Journal of clinical investigation, 121(11), 4393–4408. https://doi.org/10.1172/JCI46122

[4] Kuro-o, M., Matsumura, Y., Aizawa, H., Kawaguchi, H., Suga, T., Utsugi, T., Ohyama, Y., Kurabayashi, M., Kaname, T., Kume, E., Iwasaki, H., Iida, A., Shiraki-Iida, T., Nishikawa, S., Nagai, R., & Nabeshima, Y. I. (1997). Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature, 390(6655), 45–51. https://doi.org/10.1038/36285

[5] Kawaguchi, H., Manabe, N., Miyaura, C., Chikuda, H., Nakamura, K., & Kuro-o, M. (1999). Independent impairment of osteoblast and osteoclast differentiation in klotho mouse exhibiting low-turnover osteopenia. The Journal of clinical investigation, 104(3), 229–237. https://doi.org/10.1172/JCI5705

[6] Kawaguchi, H., Manabe, N., Miyaura, C., Chikuda, H., Nakamura, K., & Kuro-o, M. (1999). Independent impairment of osteoblast and osteoclast differentiation in klotho mouse exhibiting low-turnover osteopenia. The Journal of clinical investigation, 104(3), 229–237. https://doi.org/10.1172/JCI5705

[7] Kamemori, M., Ohyama, Y., Kurabayashi, M., Takahashi, K., Nagai, R., & Furuya, N. (2002). Expression of Klotho protein in the inner ear. Hearing research, 171(1-2), 103–110. https://doi.org/10.1016/s0378-5955(02)00483-5

[8] Nagai, T., Yamada, K., Kim, H. C., Kim, Y. S., Noda, Y., Imura, A., Nabeshima, Y., & Nabeshima, T. (2003). Cognition impairment in the genetic model of aging klotho gene mutant mice: a role of oxidative stress. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 17(1), 50–52. https://doi.org/10.1096/fj.02-0448fje

[9] Fan, J., & Sun, Z. (2016). The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells. Stem cells (Dayton, Ohio), 34(6), 1615–1625. https://doi.org/10.1002/stem.2305

[10] Jiang, W., Xiao, T., Han, W., Xiong, J., He, T., Liu, Y., Huang, Y., Yang, K., Bi, X., Xu, X., Yu, Y., Li, Y., Gu, J., Zhang, J., Huang, Y., Zhang, B., & Zhao, J. (2019). Klotho inhibits PKCa/p66SHC-mediated podocyte injury in diabetic nephropathy. Molecular and cellular endocrinology, 494, 110490. https://doi.org/10.1016/j.mce.2019.110490

[11] He, T., Xiong, J., Huang, Y., Zheng, C., Liu, Y., Bi, X., Liu, C., Han, W., Yang, K., Xiao, T., Xu, X., Yu, Y., Huang, Y., Zhang, J., Zhang, B., & Zhao, J. (2019). Klotho restrain RIG-1/NF-?B signaling activation and monocyte inflammatory factor release under uremic condition. Life sciences, 231, 116570. https://doi.org/10.1016/j.lfs.2019.116570

[12] Sahu, A., Clemens, Z.J., Shinde, S.N. et al. Regulation of aged skeletal muscle regeneration by circulating extracellular vesicles. Nat Aging 1, 1148–1161 (2021). https://doi.org/10.1038/s43587-021-00143-2

[13] Justice, J. N., Nambiar, A. M., Tchkonia, T., LeBrasseur, N. K., Pascual, R., Hashmi, S. K., Prata, L., Masternak, M. M., Kritchevsky, S. B., Musi, N., & Kirkland, J. L. (2019). Senolytics in idiopathic pulmonary fibrosis: Results from a first-in-human, open-label, pilot study. EBioMedicine, 40, 554–563. https://doi.org/10.1016/j.ebiom.2018.12.052