Targeting of Endoplasmic Reticulum Stress Signaling Pathways for the Amelioration of ANLN<sub>R431C</sub>-Induced Podocyte Apoptosis
November 07, 2019 | 10:00 AM - 12:00 PM
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Targeting of Endoplasmic Reticulum Stress Signaling Pathways for the Amelioration of ANLNR431C-Induced Podocyte Apoptosis
Genetic Diseases of the Kidney - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1002 Genetic Diseases of the Kidneys: Non-Cystic
- Hall, Gentzon, Duke University Medical Center, Durham, North Carolina, United States
- Lane, Brandon M., Duke University, Durham, North Carolina, United States
- Wu, Guanghong, Duke University, Durham, North Carolina, United States
- Chryst-Stangl, Megan, Duke Molecular Physiology Institute, Durham, North Carolina, United States
- Spurney, Robert F., Duke University Medical Center, Durham, North Carolina, United States
- Gbadegesin, Rasheed A., Duke University Medical Center, Durham, North Carolina, United States
Brandon M. Lane,
Robert F. Spurney,
Rasheed A. Gbadegesin,
We previously reported that mutations in anillin (ANLN) cause familial FSGS and that the ANLNR431C mutation induces aberrant activation of the PI3K/AKT/mTOR pathway and ER-stress-induced apoptosis in podocytes. To identify potential therapeutic targets for ANLNR431C-induced podocyte apoptosis, we sought to further delineate the ER-stress signaling pathways downstream of the PI-3K/AKT/mTOR signaling pathway.
We quantified apoptosis in our established tGFP-, ANLNWT- and ANLNR431C-overexpressing podocyte lines using the BioTek® Lionheart FX automated live cell imaging system. Biochemical pathway analyses were performed in immunoblot assays.
ER-stress signaling was activated in ANLNR431C-overexpressing podocytes at 24 hours and knockdown (KD) of CCAAT-enhancer-binding protein homologous protein (CHOP), significantly reduced ANLNR431C-induced podocyte apoptosis (p<0.0001). ANLNR431C-overexpressing podocytes showed significantly increased apoptosis relative to ANLNWT- and tGFP-overexpressing podocytes at 56 hours (p<0.0001). Pharmacologic inhibiton of mTOR and p70S6K significantly reduced ANLNR431C-induced podocyte apoptosis at 72 hours (p<0.0001). Similarly, inhibition of calcineurin phosphatase and GSK3β, two upstream regulators of CHOP expression, significantly reduced apoptosis in ANLNR431C-overexpressing podocytes relative to ANLNWT- and tGFP-overexpressing podocytes (p<0.0001). GSK3β (KD) also significantly inhibited podocyte apoptosis at 72 hours (p<0.0001). These results confirmed our prior findings. Pharmacologic inhibition of the PERK and c-Jun N-terminal Kinase (JNK) significantly reduced ANLNR431C-mediated apoptosis in podocytes (p<0.0001) highlighting the pathologic contributions of two additional signaling pathways to ANLNR431C-induced podocyte apoptosis.
These findings broaden our understanding of the pathologic role of ER-stress signaling in ANLNR431C-induced podocyte apoptosis and expand the repertoire of potential therapeutic targets for familial FSGS caused by ANLN mutations. In vivo modeling of the effects of the ANLNR431C mutation are needed to confirm the pathologic contribution of ER-stress signaling to podocyte apoptosis and to evaluate the efficacy of therapies targeting ER-stress pathways for the amelioration of ANLNR431C-induced podocyte loss.
- NIDDK Support