Fig 2: KLOTHO deficiency abrogates the anti‐renal aging effects of SGI‐1027. Mice receiving siRNA‐Control (siCtrl) or siRNA‐KLOTHO (siKl) were grouped into vehicle control, D‐gal, and SGI‐1027/D‐gal mice as before (n = 8 per group, 8 weeks). (a) Representative photomicrographs of kidney sections (Masson's trichrome staining) for siCtrl/siKl mice. The arrows indicated fibrotic collagen deposition areas. (b) Quantitation of renal fibrotic areas of (a). The effect (P) and the effect size (η2) of interaction between Kl genotype and SGI‐1027 intervention were indicated. (c) Western blots. The renal tissues from experimental mice in (a) were assayed for KLOTHO, NRF2, α‐SMA, and γH2AX. Two samples from each group were shown. (d) Quantification of (c). Data were presented as means ±SEM. *p < 0.05, three‐way ANOVA followed by Tukey's post hoc test. (e) A schematic diagram of DNMT1/3a/3b elevations and the resultant KLOTHO suppression and renal aging. Epigenetic cues upregulate DNMT1, DNMT3a, and DNMT3b, resulting in the promoter hypermethylation and expression suppressions of KLOTHO and NRF2, which accelerate D‐gal‐induced renal aging. DNA hypomethylations by DNA‐demethylating agents correct the epigenetic alterations, recover the repressions of KLOTHO and NRF2 losses, and reduce the renal aging

Fig 3: OLP preservation of KLOTHO is sensitive to gain of DNMT activities in renal cells. (a) Western blots of KLOTHO and NRF2 expression from HK2 cells treated with vehicle or D‐gal (100 mM) in absence or presence of increasing doses of OLP (20, 50, and 100 μM) or SGI‐1027 (SGI, 10 μM) for 60 h. (b) Quantifications of (a). Data are presented as means ±SEM; *p < 0.05, two‐way ANOVA. (c) qRT‐PCR of TNFA and IL6 mRNAs from HK2 cells treated with control and D‐gal (100 mM) in absence or presence of SGI (10 μM) or OLP (50 μM) for 24 h. Data were presented as box‐and‐whisker plots of 5 repeated experiments. *p < 0.05, three‐way ANOVA (d) Western blots of KLOTHO, NRF2, DNMT1, and DNMT3a from HK2 cells treated with D‐gal (100 mM) or/and OLP (50 μM) in absence or presence of DMOG (20 μM, the left panel), over‐expressed flag‐tagged DNMT1 (fDt1, the middle panel) or flag‐tagged DNMT3a (fDt3a, the right panel) for 60 h. (e) Quantification of (d). Data are presented as means ±SEM based on 5 independent experiments. *p < 0.05, two‐way ANOVA with Tukey's post hoc test