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UPMC Children’s Hospital of Pittsburgh Division of Pediatric Nephrology researchers and division chief, Carlton M. Bates, MD, secured National Institutes of Health (NIH) R01 funding in July for a new investigation to study cyclophosphamide-induced injury to the bladder urothelium, which can lead to several problems including life-threatening hemorrhagic cystitis, chronic fibrosis, and bladder cancer.
Cyclophosphamide (CPP) is an immunosuppressive chemotherapeutic agent used to treat various forms of cancer and autoimmune disorders, and it is sometimes used in the treatment of nephrotic syndrome when the condition proves to be refractory, or the patient is unable to tolerate other medications. However, CPP carries with it significant toxicity and potential to damage the bladder.
Dr. Bates’s study focuses on how a protein called fibroblast growth factor receptor 2 (FGFR2) mitigates the toxicity from CPP in the bladder. Specifically, his team has found that mice missing FGFR2 have an abnormal regenerative response in the urothelium following CPP administration. The mutant mice have prolonged injury and an inability to repair the damage to the lining of the bladder called the urothelium. What the team learns about the roles of FGFR2 will likely inform how mutations in other proteins act to mitigate the toxic effects of CPP on the bladder.
The full technical abstract of the grant is provided below.
A Robust Research Program
Dr. Bates’s new grant is the third NIH grant award to be secured by UPMC Children’s nephrology researchers in the last six months, a fact that speaks volumes as to the overall exemplary nature of the Division’s research program and faculty. In addition to Dr. Bates’s R01, Agnes Swiatecka-Urban, MD, was awarded an R01 grant to study pathogenic TGF beta activity in lung epithelial cells. Dana Fuhrman, DO, MS, another division member, was awarded a K23 grant to support her studies to identify novel biomarkers to predict the severity of acute kidney injury in patients with congenital heart disease who undergo cardiac surgery.
Cyclophosphamide (CPP)-induced injury to bladder urothelium can lead to life-threatening health conditions, including hemorrhagic cystitis and bladder cancer. The application's broad, long-term objectives are to identify mechanisms driving urothelial regeneration after CPP-induced bladder injury. CPP induces urothelial loss from apoptosis and necrosis within 48 hours, followed by proliferation and regeneration that finishes by around 28 days. Both Uroplakin-expressing cells near or at the lumenal surface and Keratin14-expressing cells in the Basal
layer of the bladder have been shown to have proliferative potential after CPP-injury. While fibroblast growth factor receptor 2 (FGFR2) is expressed throughout bladder urothelium, cell-specific roles of FGFR2 urothelial regeneration after CPP-injury are unknown.
Preliminary data show that mice with conditional deletion of Fgfr2 in all bladder urothelial layers have defective urothelial repair after injury. At three days post-CPP, controls had urothelial hyperplasia, significant restoration of Uroplakin staining and major resolution of inflammation and hemorrhage, while mutant bladders had less hyperplasia, attenuated Uroplakin staining, and ongoing hemorrhage and inflammation. While both mutants and controls had expansion of Keratin 14-expressing cells across Basal layers three days after CPP, mutant Keratin 14+ cells were hypertrophic with enlarged nuclei suggesting a cell cycle defect. Cell cycle profiling and assays for DNA content suggest that mutant Keratin 14+ cells have aberrant endoreplication (DNA replication without completion of mitosis, leading to polyploidy). Fgfr2- mutant cells undergoing apparent endoreplication also had evidence of increased DNA damage/replication stress three days after injury.
Given that FGFR2 stimulates Erk that can suppress cell cycle entry and endoreplication, Erk and its readouts were assessed, and both were reduced in mutants three days after CPP. Regeneration defects, including apparent endoreplication in Keratin 14+ cells, persisted 10 days after CPP. Together, the hypothesis is that endogenous FGFR2 signaling promotes regeneration after CPP by repressing endoreplication in Keratin 14-expressing urothelial cells via Erk.
To test this hypothesis, the following aims are proposed.