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Division of Pediatric Nephrology Researcher Obtains New R01 Grant to Study Bladder Injury; Pediatric Insights Nephrology Fall 2019

November 15, 2019

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 immuno-suppressive 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’ 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.

Technical Abstract

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 Keratin 14-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.

Aim 1: Determine how FGFR2 promotes regeneration of bladder urothelium after CPP-injury. The hypothesis is that FGFR2/ERK signaling suppresses pathological endoreplication and promotes regeneration after CPP injury. Whole animal and cell-based assays will elucidate roles of FGFR2/ERK
to suppress endoreplication and drive regeneration, including genetic rescue with constitutively active ERK in mutants and
chemical rescue to accelerate repair in controls.

Aim 2: Identify cell-specific roles of FGFR2 signaling in the bladder urothelium in regeneration after CPP injury. The hypothesis is that FGFR2 acts in Keratin 14+ Basal cells to promote regeneration after CPP injury. FGFR2 will be deleted specifically in Uroplakin-expressing and Keratin 14-expressing urothelial layers to identify cell-specific actions after CPP-injury.

A Robust Research Program

Dr. Bates’ new grant is the fourth NIH award to be secured by UPMC Children’s nephrology researchers in the last six months, a fact that speaks volumes about the exemplary nature of the Division’s research program and faculty. In addition to Dr. Bates’ 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. Sunder Sims-Lucas, PhD, secured a new R56 award to continue his acute kidney injury studies and the possible protective role that a family of genes called the sirtuins may play in AKI.

Dr. Bates started the course in 2017, which is a part of the SPR Peer Mentoring Group.

Dr. Bates’ course begins with an initial face-to-face meeting with the selected applicants at the annual spring Pediatric Academic Societies (PAS) Meeting. Dr. Bates and the participants then have monthly teleconferences in which he provides guidance for how to write individual grant sections. The participants then submit grant section drafts for review by Dr. Bates and a peer, after which Dr. Bates and the peers provide oral and written critiques at a follow-up meeting. Two classes of participants have completed the course to date and increasing numbers of Junior Section Members are applying to take the course each year. The SPR also is tracking past participants’ successes in obtaining K awards.

Dr. Bates intends to continue the course after he completes his term as a SPR Council member in 2019.

More About Dr. Bates

Dr. Bates received his medical degree from The Ohio State University College of Medicine and completed a residency in pediatrics at Nationwide Children’s Hospital. He then completed a fellowship in pediatric nephrology at the University of Texas Southwestern (UTSW) Medical Center in Dallas.

After one year as nontenure track faculty at UTSW, Dr. Bates became an assistant professor of pediatrics at The Ohio State University/Nationwide Children’s Hospital in 1999. He was tenured and promoted to associate professor in 2007. In 2008, Dr. Bates accepted the position of division chief of pediatric nephrology and the director of the pediatric nephrology fellowship program UPMC Children’s Hospital of Pittsburgh and the University of Pittsburgh School of Medicine. Dr. Bates was promoted to professor of pediatrics in 2014. In 2016, Dr. Bates assumed the role of Vice Chair of Basic Research in the Department of Pediatrics at the University of Pittsburgh School of Medicine.

Dr. Bates’ research is focused on the role of fibroblast growth factor receptors (FGFRs) and their adapter binding proteins in kidney and lower urinary tract development and disease in murine models. Utilizing conditional and global knockout approaches to manipulate expression of FGFRs 1 and/or 2 and their adapter proteins, his lab has uncovered many novel roles for the receptors in multiple lineages and at different stages of kidney and lower urinary tract development. These murine lines often mimic many of the congenital forms of kidney and bladder disease that are leading causes of renal and lower urinary tract disease in children. Recently, the lab has identified novel roles for FGFR2 in protecting against bladder urothelial injury and driving early regeneration of outer urothelial layers after cyclophosphamide infusion.

Dr. Bates was elected to the Society for Pediatric Research in 2002 and was elected to the SPR Council in 2016. Since beginning his tenure on the SPR Council, Dr. Bates has been a member of the Mentoring Work Group and the Finance Committee. He also is actively participating in writing an SPR perspectives manuscript that will offer strategies for early stage pediatric clinician-scientists (and their institutional leadership) to increase their chances of success in obtaining external research funding.