Dixon Lab

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    + DNA Damage and Response in the Bladder Microenvironment

    Children born with complex urogenital anomalies often require reconstructive surgery called bladder augmentation using a patch of gastrointestinal tissue to provide a more functional bladder to protect the native kidneys or a kidney transplant from high bladder pressures. An uncommon but frequently fatal complication of these surgeries is cancer of the gastrointestinal tissue in the bladder, but the cause is largely unknown. The specific aim of this project is to study the DNA damage response and DNA repair of gastrointestinal and bladder cells in the bladder microenvironment, both in cell culture and in an animal model of bladder augmentation.

    Funding Source: NIH / NIDDK

    This bladder epithelial cell was subjected to gel electrophoresis after incubation with 50 µM etoposide. The tail area (area under the blue curve) is indicative of DNA double strand breaks induced by etoposide.  Hyperosmolality appears to disrupt the ability of some cell types in detecting and repairing DNA damage.

    This bladder epithelial cell was subjected to gel electrophoresis after incubation with 50 µM etoposide. The tail area (area under the blue curve) is indicative of DNA double strand breaks induced by etoposide.  Hyperosmolarity appears to disrupt the ability of some cell types in detecting and repairing DNA damage.

    + Role of the Primary Cilium in Osmotic Response of Renal Epithelial Cells

    Primary cilia are microtubule-based structures that originate from a basal body at the centrosome (or microtubule organizing center) and project from the surface of epithelial and many other cell types. Primary cilia serve as sensory organelles of the cell, detecting chemical stimuli including the osmolality of their environment. These stimuli are transduced to cell signaling pathways influencing cellular differentiation, proliferation, survival, and migration. The specific aim of this project is to assess the dependence of renal epithelial cells upon primary cilia in sensing osmolality, and the components of this osmosensory mechanism.

    Funding Source: NIH / NIDDK

    Collaborators: Nancy Kleene, PhD, University of Cincinnati; Steve Kleene, PhD, University of Cincinnati; P. Darwin Bell, PhD, Medical University of South Carolina

    TRP channel localization on the primary cilium in renal epithelial cells.  Immunofluorescence of acetylated α-tubulin demonstrating the primary cilium (green, left panel) and a TRP channel (red, middle panel).  Co-localization of the TRP channel with acetylated α-tubulin in a merged image (yellow, right panel) indicates localization on primary cilia.

    TRP channel localization on the primary cilium in renal epithelial cells. Immunofluorescence of acetylated α-tubulin demonstrating the primary cilium (green, left panel) and a TRP channel (red, middle panel). Co-localization of the TRP channel with acetylated α-tubulin in a merged image (yellow, right panel) indicates localization on primary cilia.

    + Cell Signaling Pathways of Renal Cysts and Angiomyolipomas in TSC

    Altered cellular proteostasis, including conditions such as endoplasmic reticulum (ER) stress, may contribute to the pathogenesis of renal angiomyolipomas and cysts in Tuberous Sclerosis Complex (TSC). ER stress describes accumulation of misfolded proteins in the ER lumen, to which the cell responds by initiating an adaptive mechanism, the unfolded protein response (UPR), to counteract the imbalance or initiate apoptosis if ER stress is prolonged. In TSC, mutations in TSC1 or TSC2 deregulate activity of the mammalian target of rapamycin complex 1 (mTORC1), which results in constitutive protein translation. Our laboratory recently found that TSC2-deficienthuman renal angiomyolipoma cells experience elevated ER stress, and are more sensitive to treatments that exacerbate ER stress, than TSC2-sufficient counterparts. We also determined that ER stress is elevated in human TSC patient-derived renal angiomyolipoma and cyst tissue. The goal of this project is to determine whether altered proteostasis, such as ER stress, can be leveraged therapeutically for treatment of TSC renal disease.

    Funding Source: Tuberous Sclerosis Alliance

    Collaborators: Julie Yin, MD, Children’s Hospital of Atlanta

    Enhanced sensitivity of human renal angiomyolipoma cells to ER stress.

    Enhanced sensitivity of human renal angiomyolipoma cells to ER stress.  Human renal angiomyolipoma cells carrying a mutation in TSC2 (TRI102, panels "A" and "C") are more sensitive to induction of endoplasmic reticulum (ER) stress by treatment with the proteasome inhibitor MG-132, as indicated by enhanced nuclear expression of CHOP in panel "C", than cells in which TSC2 has been re-expressed (TRI103, panels "B" and "D"). From Siroky et al. AJP Renal, 2012.

    + Role of the Primary Cilium in Polycystic Kidney Diseases including TSC

    Defects in primary cilia are associated with most forms of inherited renal cystic disease. Many human ”cystoproteins” are now known to localize to the primary cilium or centrosome, including the Tuberous Sclerosis Complex (TSC) proteins hamartin and tuberin. Our laboratory has recently found that elevated mammalian target of rapamycin complex 1 (mTORC1) activity due to deficiency of tuberin in renal cystic cells also affects cilia expression. The goal of this project is to identify the mechanism(s) whereby cilia disruption contributes to TSC renal cystic disease.

    Funding Source: Tuberous Sclerosis Alliance

    Collaborators: Nancy Kleene, PhD, University of Cincinnati; P. Darwin Bell, PhD,Medical University of South Carolina