F: H&E-stained bladder displays some sloughing of urothelial cells (arrow). response to damage, peaking at 3 times in both treatment groupings; however, proliferation prices had been low in the KGF group at 3 times, consistent with much less injury. Three times after damage, unlike handles, KGF-pretreated mice acquired regenerated superficial cells. At 10 and 28 times after cyclophosphamide treatment, KGF-pretreated mice acquired small proliferation and proclaimed recovery of urothelial levels, whereas the phosphate-buffered saline group acquired ongoing regeneration. Administration of KGF to uninjured mice reproduced ERK-driven KRT5+/KRT14? proliferation observed in harmed mice; KRT14+ cells had been unaffected. KGF pretreatment blocks cyclophosphamide-induced basal and intermediate cell apoptosis, most likely by phosphorylated AKT, and drives phosphorylated ERKCmediated KRT5+/KRT14? cell proliferation, resulting in early urothelial regeneration. Cyclophosphamide, an alkylating agent that’s employed for solid tumors broadly, leukemias, lymphomas, and nononcologic illnesses, such as for example lupus, is extremely bladder dangerous.1 Cyclophosphamide is metabolized to acrolein, which concentrates in the urine, resulting in direct harm to bladder urothelium. Clinically, severe toxicity is certainly manifested as hemorrhagic cystitis, which runs in intensity from microscopic hematuria and/or unpleasant lower urinary system symptoms to serious life-threatening bleeding. Persistent sequelae include bladder contractures and fibrosis. Sufferers treated with cyclophosphamide for either oncologic or nononcologic disease also?possess dose-dependent increased dangers of developing urothelial cancers,2, 3, 4, 5 with 1 research acquiring a nearly 15-fold risk for lymphoma survivors provided 50 g cumulative dosages.6 Current therapies to prevent hemorrhagic cystitis are?only?partially efficacious.1,7,8 Mesna (sodium-2-mercaptoethanesulfonate), the most commonly used therapy, directly binds acrolein in the urine; however, it may cause hypersensitivity reactions and often does not prevent cystitis. One study found that 66% of patients treated with mesna to prevent injury from ifosfamide (metabolized to acrolein, like cyclophosphamide) had urothelial injury by cystoscopy.9 Hydration, another commonly used therapy to dilute acrolein, is also at best partially efficacious and may be contraindicated in patients who cannot tolerate fluid overload.8 Antioxidants or anti-inflammatory drugs have no superior efficacy to mesna.8 There are also no data that mesna (or any drug) prevents cyclophosphamide-driven urothelial cancer.10 Many preclinical and clinical studies have shown that fibroblast growth factor receptor 2 (FGFR2) signaling reduces injury and drives regeneration in epithelial tissues after noxious stimuli.11, 12, 13, 14, 15, 16, 17 FGFR2 is a receptor tyrosine kinase, which has a IIIb isoform that is found in epithelium, including urothelium, and that binds to ligands, including FGF7 [alias keratinocyte growth factor (KGF)].18 Exogenous recombinant human KGF ameliorates toxin or radiation-mediated injury in oral, retinal, alveolar, and intestinal epithelia.12, 13, 14, 15, 16, 17 In 1997, Ulich et?al11 published that one i.v. dose of KGF in rats 24 hours before cyclophosphamide administration led to increased urothelial proliferation versus vehicle-pretreated mice and USL311 led to marked improvement in bladder histology. The report did not reveal whether the beneficial effects of KGF were related to the hyperproliferation (ie, accelerated regeneration) and/or cytoprotection and did not explore mechanisms of KGF activity in the bladder. Our study examines a detailed histologic analysis of cyclophosphamide-induced injury, how KGF acts to deliver urothelial cytoprotection and drive proliferation, and the mechanisms underlying the latter actions. Materials and Methods Mice Female FVB/NJ mice (aged 2 to 3 3 months) were used for all experiments. All the proposed mouse experiments were approved by the University of Pittsburgh (Pittsburgh, PA) Institutional Animal Care and Use Committee in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. Cyclophosphamide Injections Mice were administered USL311 either 150 mg/kg USL311 i.p. cyclophosphamide (Sigma-Aldrich, St. Louis, MO; catalog number C7397) dissolved in phosphate-buffered saline (PBS) or i.p. PBS alone (sham injury). KGF Pretreatment Mice were administered 5 mg/kg s.c. injections of KGF (R&D Systems, Minneapolis, MN; catalog number 251-KG-010) dissolved in PBS or of PBS alone (vehicle), 24 hours before cyclophosphamide (or PBS) injections. In some instances, no cyclophosphamide (or i.p. PBS) was administered after KGF or PBS. Histology, IF, and USL311 TUNEL Assays For histology, paraffin-embedded tissues were sectioned (6?m thick) and stained with hematoxylin Gja7 and eosin (H&E). For immunofluorescence (IF), dewaxed paraffin-embedded sections were subjected to antigen retrieval in a pressure cooker for 15 minutes in Tris-EDTA, pH 9.0, buffer and then blocked with normal donkey serum for 1 hour at room temperature. Sections were then incubated overnight at 4C with the following primary antibodies: antiCkeratin 20 (KRT20) at 1:50 (Agilent Technologies, Santa Clara, CA; catalog number M7019; RRID:AB_2133718), keratin 5 (KRT5) at 1:200 (BioLegend, San Diego, CA; catalog number 905901) (RRID Portal, Apoptosis Fluorescein Detection kit (EMD Millipore, Burlington, MA; catalog number S7111-kit), according to manufacturer’s instructions. Slides were imaged with a Leica DM2500 fluorescence microscope (Leica Microsystems, Buffalo Grove, IL) or a Zeiss LSM 710 confocal microscope (Carl Zeiss, Thornwood, NY). Total urothelial proliferation rates were assessed by counting Ki-67+/all DAPI+ cells and individual cell type proliferation rates were assessed.