Oral Therapies Boost Alpha-Klotho, Key IPF Protective Protein, in Mice

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by Steve Bryson, PhD |

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Oral therapies that kill aging cells were found to boost the levels of alpha-Klotho, a protective protein that declines with age and safeguards animals against age-related diseases — including idiopathic pulmonary fibrosis (IPF) — in a study involving older mice.

So-called senolytic therapies are designed to kill aging senescent cells, or those that no longer divide or grow. The accumulation of senescent cells in the body is associated with aging, and such cells are thought to contribute to age-related diseases.

The researchers noted that levels of the protective alpha-Klotho protein also were elevated in the urine of IPF patients who participated in a recent Phase 1 pilot study. That study showed that combined treatment with two senolytic agents — dasatinib and quercetin — improved physical function.

The new findings support the development of orally-active small molecules to treat IPF by increasing alpha-Klotho, which also may be a helpful biomarker for senolytic therapy activity in clinical trials, the researchers noted.

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“We show that there is an avenue for an orally active, small-molecule approach to increase this beneficial protein and also to amplify the action of senolytic drugs,” James Kirkland, MD, PhD, the study’s senior author, at the Mayo Clinic, in Minnesota, said in a press release.

The study, “Orally-active, clinically-translatable senolytics restore α-Klotho in mice and humans,” was published in the journal eBioMedicine.

Studies show senescent cells release molecules that promote chronic inflammation and tissue scarring, two hallmarks signs of IPF. Further, senescence markers are more abundant in IPF patients than in healthy individuals. Researchers note that older age also is associated with a higher risk of IPF, in which scarring or fibrosis in the lungs makes it harder for patients to breathe.

As senescent cells increase, a protective protein called alpha-Klotho declines. Mice deficient in alpha-Klotho exhibit accelerated aging, whereas overproducing this protein increases lifespan. In IPF, mice with induced lung fibrosis have lower alpha-Klotho levels, and mice modified to overproduce alpha-Klotho are protected from induced lung fibrosis.

These findings suggest that boosting alpha-Klotho holds therapeutic potential, but current methods are not translatable to the clinic.

Kirkland and colleagues wondered if alpha-Klotho and cellular senescence were related and, if so, whether treatment with orally-available small molecules called senolytics, which trigger the death of senescent cells, is a clinically effective way to increase the protective alpha-Klotho protein.

First, researchers exposed three primary human cell lines — from the kidney, brain, and umbilical cord veins — to radiation to create senescent cells. When the cell media used to propagate senescent cells was added to non-senescent cells, alpha-Klotho expression (production) was reduced.

These reductions were partially prevented by adding antibodies that target two proteins associated with cell senescence, activin A and Interleukin 1 alpha (IL-1 alpha), also referred to as senescence-associated secretory phenotype (SASP) factors. Conversely, treating non-senescent cells with activin A and IL-1 alpha caused a decrease in alpha-Klotho expression.

Then, when researchers transplanted senescent or non-senescent fat cell precursors into young mice, they found that alpha-Klotho was reduced in the urine and brain of animals receiving senescent cells compared with mice transplanted with non-senescent cells.

“We also are first to link the potential impact of fat-resident senescent cells on brain [alpha]-Klotho,” said Yi Zhu, PhD, the study’s first author and a Mayo Clinic physiologist and biomedical engineer.

“This may open another avenue to investigate the impact of peripheral senescent cells on brain aging,” Zhu said.

The team then assessed the effect of removing cells that express the p16Ink4a gene, which is increasingly active as cells age and is associated with cell senescence. Alpha-Klotho levels from urine, kidneys, and brain, which were lower in older mice than younger mice, were increased when these cells were removed.

In addition, kidney activin A and brain IL-1-alpha were high in old mice, but lower after p16Ink4a removal, “supporting the hypothesis that senescent cells contribute to the age-related decline in [alpha]-Klotho in part through the SASP factors, activin A and [IL-1-alpha],” the researchers wrote.

Next, they demonstrated that treating aged mice orally with the senolytic agents dasatinib plus quercetin, or fisetin, increased the levels of alpha-Klotho in the urine. In young mice with diet-induced obesity, dasatinib plus quercetin effectively raised urinary alpha-Klotho. These mice had an increased number of senescent cells compared with lean mice, and low levels of urinary alpha-Klotho.

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In young mice transplanted with senescent cells, dasatinib plus quercetin or fisetin also increased urinary alpha-Klotho.

In the brains of old mice, in which alpha-Klotho decreases with age, treatment with senolytics also increased alpha-Klotho levels. The same was seen in young obese mice. In untreated obese mice, low levels of alpha-Klotho were related to the presence of more senescent fat cells.

Additional experiments showed that treatment with senolytics did not increase alpha-Klotho in cultured non-senescent cells nor in the urine of young mice.

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

Lastly, Kirkland and colleagues had previously reported that treating IPF patients with dasatinib plus quercetin over three weeks improved physical function, with increased walking distance and speed, and enhanced ability to stand up from a chair. Those findings had come from a small Phase 1 pilot study (NCT02874989)

An analysis of urine from these study participants showed that alpha-Klotho increased following treatment, and that this increase was correlated with a decrease in urinary SASP factors.

“Senolytics appear to be an effective small molecule, orally-active interventional strategy for increasing [alpha-Klotho],” the authors wrote.

Testing alpha-Klotho in urine “could be a useful approach for monitoring effects of senolytics during clinical trials or, eventually, if senolytics are translated into clinical practice, in the course of treating patients,” they wrote.