Abstract
Purpose :
Klotho is a transmembrane glycoprotein primarily expressed in renal distal convoluted tubules. Klotho knockout mice show premature aging whereas klotho over-expressing mice have extended life span. The extracellular domain (ECD) of klotho can be cleaved. This soluble form, containing amino acid (AA) sequence 34-981, circulates in blood and has endocrine functions. The soluble klotho has been shown to have anti-inflammatory, antioxidant, and senolytic effects on cardiac and nervous tissue. Aging and inflammation are major risk factors for dry eye disease. Therefore, the present study was designed to test the senolytic effect of recombinant soluble klotho-α (ECD 34-981 AA) on human corneal epithelial cells.
Methods :
Senescence was induced in cultured human corneal epithelial cells using doxorubicin. The onset of senescence was confirmed by induction of β-galactosidase (gal) and a decrease in proliferation measured by EdU and the β-gal immunostaining and quantified using flow cytometry and confocal microscopy. CDKN1A (p21) levels were quantified using qRT-PCR. The experimental design included control cells +/- klotho (50 ng/ml) treatment and doxorubicin-exposed senescent cells +/- klotho. All the experiments were conducted n=6.
Results :
Our data demonstrates that klotho treatment significantly prevented doxorubicin-induced senescence as demonstrated by restoration of cellular proliferation, attenuation of β-gal expression, and inhibition of p21 induction. The quantification data shows that doxorubicin treatment reduced proliferation by 70%, and klotho treatment significantly prevented this decrease in proliferation by 40%. Furthermore, 71% of doxorubicin-exposed cells showed β-gal staining whereas only 45% of klotho-treated cells showed β-gal staining. Lastly, p21 levels were increased by 1.2-fold in doxorubicin-exposed cells, and klotho rescued this increase back to the control levels.
Conclusions :
Klotho treatment has senolytic effect on human corneal epithelial cells and could potentially be a novel approach to treat age-related dry eye. Further in vivo studies in aged mice are warranted.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.