FIGURE 3.

Transdifferentiation and recruitment of ageing REP cells. A, Example of αSMA immunofluorescence (IF; green) combined with REP cell tagging in Epo‐Cre^ERT2#1xtdTomato mice (red) and nuclear staining by DAPI (blue). B, Kidneys were excised at the indicated time points after the 4 hours 0.1% CO stimulus and the labelled reporter cells were automatically counted. The number of analysed cells derived from 8 mice per timepoint is indicated. C, Example of Epo mRNA fluorescence in situ hybridization (FISH; green) combined with immunofluorescence detection of tdTomato in tagged REP cells by an anti‐red fluorescent protein (RFP) antibody (red) and nuclear staining by DAPI (blue). Epo mRNA/tdTomato double‐positive REP cells are depicted in yellow. D and E, Quantification of tdTomato/Epo mRNA double‐positive cells. The numbers of analysed cells derived from 5 to 8 mice per timepoint are indicated. F, Schematic illustrating the conditional tagging of REP reporter cells followed by a second hypoxic stimulus after the indicated time periods. G, Detection of Epo mRNA/tdTomato double‐positive REP cells as in (C). H and I, Quantification of tdTomato/Epo mRNA double‐positive cells. The numbers of analysed cells derived from 5 to 11 mice per timepoint are indicated. B, D, E, H, I, All data are shown as average + SEM. One‐way ANOVA followed by Dunnett’s post‐hoc correction versus the corresponding values of week 1 was used to statistically evaluate cell compositions (*P < .05)