Figure 2.

Representative images showing effects of keratinocyte growth factor (KGF) pretreatment 6 hours and 1 day after cyclophosphamide injection. A–J: Images from 6 hours after cyclophosphamide administration. A and F: Hematoxylin and eosin (H&E) staining shows PBS-pretreated bladders have extensive urothelial injury, lumenal debris, and cellular sloughing (arrowhead, A) with few remnant urothelial cells attached (arrow, A) versus KGF-pretreated bladders that have loss of outer superficial cells (arrowhead, F) and preservation of deeper urothelial layers (arrow, F). B and G: KRT20 immunofluorescence (IF; red) in sections adjacent to A and F, respectively, shows many of the sloughing cells in PBS-pretreated (B) and KGF-pretreated (G) bladders are KRT20⁺ (arrowheads). C and H: Uroplakin 3a (UPK3) IF (white) in sections adjacent to B and G, respectively, shows PBS-pretreated bladders have many sloughing UPK3⁺ cells (arrowhead, C) with few UPK3⁺ intermediate cells adherent to the bladder (arrow, C); KGF-pretreated bladders also have UPK3⁺ sloughing cells (arrowhead, H) but many UPK3⁺ intermediate cells remain attached (arrow, H). D and I: KRT5 IF (green) in the same sections as C and H, respectively, shows that PBS-pretreated bladders have only a single layer of remaining KRT5⁺ cells that are exposed to the lumen (arrow, D), whereas KGF-pretreated bladders have many layers of apparently uninjured KRT5⁺ cells (arrow, I). E and J: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (green) in sections adjacent to D and I, respectively, shows apoptosis in many PBS-pretreated urothelial cells (arrowheads) but none in KGF-pretreated bladders (J), including in sloughing superficial cell nuclei (arrows). B–E and G–J: Blue indicates DAPI. E and J: K–T: Images from 1 day after cyclophosphamide administration. K and P: H&E staining shows that PBS-pretreated bladders (K) have individual urothelial cells (short arrow) and sheets of cells (long arrow) sloughing, leaving regions of denuded bladder (arrowhead) with hemorrhage and inflammation (concave arrows), whereas KGF-pretreated bladders (P) appear to have minimal urothelial damage (concave arrowhead). L and Q: KRT20 IF (green) in sections adjacent to K and P, respectively, is negative (arrowheads) in both PBS-pretreated (L) and KGF-pretreated (Q) bladders, showing ongoing absence of superficial cells in both. M and R: UPK3 IF (white) in sections adjacent to L and Q, respectively, shows that the PBS-pretreated bladders (M) have UPK3⁺ intermediate cells within the sheet of sloughing cells (long arrow) and few remaining UPK3⁺ cells remaining attached to the bladder (short arrow), whereas the KGF-pretreated bladders (R) have robust and continuous UPK3 expression across the lumenal surface of the urothelium (arrowhead), consistent with intact UPK3⁺ intermediate cells. N and S: KRT5 IF (green) in the same sections as M and R, respectively, shows that the PBS-pretreated bladders (N) have KRT5⁺ intermediate/basal cells within the sheet of sloughing cells (arrow) and at most a single layer of KRT5⁺ cells remaining attached to the bladder (short arrow), whereas the KGF-pretreated bladders (S) have robust and continuous KRT5 expression across the deeper regions of the urothelium (arrowhead), consistent with intact KRT5⁺ intermediate and basal cells. O and T: TUNEL staining (green) in sections adjacent to N and S, respectively, shows sheets of apoptotic cells in PBS-pretreated urothelium (arrowheads; O) and no apoptosis in KGF-pretreated (T) bladders. L–O and Q–T: Blue indicates DAPI. Dotted lines indicate demarcation between urothelial layer and underlying lamina propria. Scale bars = 50 μm (A–T). L, lumen.