Fig. 3. Rac1 promotes proliferation and normal mitotic progression during repair.

(A) Optically cleared full medullary slices of baseline, obstructed (“UUO”), and unobstructed reversed Rac1^f/f and AQP2:Rac1^f/f kidneys. Ki-67 signal inside AQP2⁺ CDs was filtered and converted to white dots. Scale bars, 100 μm. (B) Quantification of (A) showing Ki-67–positive cells per 100-μm-long CD segment with each dot representing individual mice. n = 4 mice per group. (C) Apoptosis (TUNEL; white) labeling of CDs (AQP2⁺; magenta) of paraffin kidney sections in reversed Rac1^f/f and AQP2:Rac1^f/f mice. (D) Quantification of TUNEL-positive cells in CDs per medullary HPF with each dot representing individual samples. n = 4 mice per group. (E) Representative flow cytometry plots of AQP2⁺ CD cells of reversed Rac1^f/f and AQP2:Rac1^f/f kidneys showing side scatter (SSC-A) against DNA [4′,6-diamidino-2-phenylindole (DAPI)] with a G₂-M phase–specific cell cycle gate and corresponding subpopulation percentage shown in the plot. (F) Quantification of G₂-M phase–specific cell populations as shown in the gating in (E) with four mice (dots) per group; bars are means ± SD. (G) Mitotic (pH3-positive; green) metaphase cells (condensed, aligned DNA) of repairing (reversed) CDs (AQP2; magenta) of Rac1^f/f and AQP2:Rac1^f/f mice were analyzed using 3D super-resolution confocal imaging. The far left column shows orthogonal Z-slices (scale bars, 2.5 μm; yellow continuous line outlines the apical lumen) as indicated by the yellow dashed line in the cross section in the middle column (scale bars, 5 μm). The far right column depicts insets as outlined by a red continuous box (scale bars, 2.5 μm). (H) Relative distribution of metaphase abnormalities based on a morphological assessment as shown in (G). At least 10 mitoses were analyzed per group. *P < 0.05.