Fig. 6. By targeting senescence, FOXO4-DRI counters loss of renal function of Xpd^TTD/TTD mice.

A) Quantification of renal filtering capacity measured by plasma [Urea] in 13, 26 and 130w old wildtype mice and 13 and 26w Xpd^TTD/TTD mice. B–D) Visualization of senescence (SA-β-GAL), the major SASP factor IL-6 and FOXO4 foci in 26w + 130w wildtype and 26w Xpd^TTD/TTD old kidneys. Tubuli (T), Glomeruli (G). Inset C): Magnification of SA-β-GAL to reveal affected areas. Inset D) quantification of the % of renal cells expressing ≥10 FOXO4 foci. E) TUNEL assay to detect apoptosis in kidney sections of 130w old wt mice treated 3d with PBS or FOXO4-DRI. See Fig. S6A–D for pipeline and results with shFOXO4. F) Quantification of the % of platelets at time of sacrifice vs. baseline for wt and Xpd^TTD/TTD mice treated with PBS or FOXO4-DRI. See also Fig. S6E. G) Representative Images of kidneys from 26w wt or Xpd^TTD/TTD mice stained for LMNB1 loss. Quantified are the average number of nuclei per kidney positive for LMNB1 (at least 400nuclei per mouse). H) Viability assay on control or senescent IMR90 incubated with recombinant IL1α, IL1β or IL1 receptor antagonist (IL1-RA) 24h prior to exposure of FOXO4-DRI. I) Viability plot showing the effect of FOXO4-DRI on control and senescent IMR90 pretreated with Cortisol and LPS, prior to FOXO4-DRI treatment. J) Staining as in G), but for the SASP marker IL-6. Quantified is the average IL-6 intensity per kidney over at least 3 frames per mouse for at least 4 mice per group. K) Quantification of the % plasma [Urea] of three pooled cohorts of wt and Xpd^TTD/TTD mice (n=7–8 mice/treatment) after 30d treatment with PBS or FOXO4-DRI. Data are represented as mean +/− SEM. See also Fig. S4G. L) Experiment as in K), but using Ganciclovir (GCV) to mediate semigenetic clearance of senescent cells through the Thymidine Kinase expressed by the p16∷3MR construct. As GCV is i.p. administered, also FOXO4-DRI was i.p. administered in this experiment.