Figure 8.

Representative images and graphs of urothelial proliferation in keratinocyte growth factor (KGF)–pretreated bladders 10 days after cyclophosphamide injection. A and F: Hematoxylin and eosin staining shows that PBS-pretreated bladders (A) have evidence of ongoing cellular hyperplasia with up to seven to eight layers of smaller cells throughout the urothelium (arrow, A) compared with the KGF-pretreated bladders (F) that have three to four cell layers (arrow, F) and many large superficial cells (arrowheads), similar to uninjured urothelium. B and G: Immunofluorescence (IF) for KRT20 (red) in sections adjacent to A and F, respectively, shows the return of some signal in the outer layers of the PBS-pretreated urothelium (arrowhead, B), although gaps without KRT20 expression remain (concave arrowhead) compared with KGF-pretreated bladders (G) that have robust and continuous KRT20 staining across the lumenal surface (arrowhead, G), consistent with the return of mature superficial cells (arrows in B and G illustrate the larger numbers of DAPI⁺ urothelial cells in the PBS group versus the KGF group, respectively). C–E and H–J: Triple-label IF for Ki-67 (red), KRT14 (white), and KRT5 (green) in PBS-pretreated (C–E) and KGF-pretreated (H–J) bladder sections adjacent to B and G, respectively. C and H: Ki-67 staining shows that the PBS group (C) continues to have robust numbers of proliferating (Ki-67⁺) urothelial cells, whereas the KGF group (H) has few proliferating cells. B, C, G, and H: Blue indicates DAPI. Dotted lines indicate demarcation between urothelial layer and underlying lamina propria. D and I: Ki-67 and KRT14 merged staining shows that in PBS-pretreated mice (D), most of the proliferating (Ki-67⁺) urothelial cells are also KRT14⁺ cells (arrowheads, D) that remain spread across the urothelium and few Ki-67⁺ cells are KRT14⁻ (concave arrowhead, D); in contrast, KGF-pretreated bladders (I) have few KRT14⁺ cells and among the few Ki-67⁺ cells, similar numbers are KRT14⁺ (arrowhead, I) and KRT14⁻ (concave arrowhead, I). E and J: Ki-67 and KRT5 merged staining shows that most of Ki-67⁺ proliferating cells in both the PBS group (E) and the KGF group (J) are KRT5⁺, including in KRT14⁺ (arrowheads) and KRT14⁻ (KRT5⁺ only; concave arrowheads) cells (arrow in J shows a Ki-67⁺/KRT5⁻ cell with a large nucleus, likely representing a polyploid superficial cell). K: Graph showing significantly higher percentages of proliferating cells (Ki-67⁺/DAPI⁺ cells) in the PBS-pretreated urothelium versus KGF-pretreated urothelium 10 days after cyclophosphamide injection. L: Graph showing that the percentages of KRT14⁺ proliferating (Ki-67⁺) urothelial cells are much higher in the PBS- versus KGF-pretreated mice [and that the small number of proliferating KRT14/KRT5⁻ (no label) cells are also statistically higher in the PBS group], whereas the percentages of proliferating KRT5⁺/KRT14⁻ (KRT5 only) cells are not statistically different. Data are expressed as means ± SEM (L). N = 6 (2 planes per bladder and 3 bladders per group; L). **P < 0.01, ***P < 0.001. Scale bars = 50 μm (A–J). L, lumen.