Abstract: TH-PO1066
Evaluation of the Human FSGS-Inducing ANLN R431C Variant in CRISPR-Cas9 Mice
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Lane, Brandon M., Duke University School of Medicine, Durham, North Carolina, United States
- Wu, Guanghong, Duke University School of Medicine, Durham, North Carolina, United States
- Chryst-Stangl, Megan, Duke University School of Medicine, Durham, North Carolina, United States
- Hall, Gentzon, Duke Molecular Physiology Institute, Durham, North Carolina, United States
- Spurney, Robert F., Duke University School of Medicine, Durham, North Carolina, United States
- Barisoni, Laura, Duke University School of Medicine, Durham, North Carolina, United States
- Miller, Sara E., Duke University School of Medicine, Durham, North Carolina, United States
- Howell, David, Duke University School of Medicine, Durham, North Carolina, United States
- Gbadegesin, Rasheed A., Duke University School of Medicine, Durham, North Carolina, United States
Background
We previously reported that the ANLN R431C variant causes focal segmental glomerulosclerosis (FSGS) and induces endoplasmic reticulum (ER) stress induced apoptosis in cultured human podocyte cells. To further understand the molecular mechanisms underlying disease, we examined the phenotypic effects of this variant in vivo using a CRISPR-Cas9 generated mouse model.
Methods
We generated an orthologous ANLN R431C knock-in point mutation in mice (R426C) using CRISPR-Cas9 technology. ANLN+/+, ANLNR426C/+, ANLNR426C/R246C mice were challenged with a sub-therapeutic dose of nephrotoxic antibodies at 22 weeks and evaluated for 5 weeks post injection. Kidney sections were evaluated by two independent pathologists, blinded to genotype, through PAS staining and electron microscopy.
Results
When challenged with nephrotoxic antibodies in a kidney disease resistant genetic background, ANLNR426C/R246C mice displayed increased proteinuria compared to ANLNR426C/+ (p=0.049) and ANLN+/+ (p=0.0064) mice. Light microscopy evaluation of ANLNR426C/R246C kidney sections revealed increased protein casts (p<0.0001), as well as larger (p=0.018) and more sclerotic (p=0.0005) glomeruli when compared to wildtype littermates. Semi-quantitative analysis using electon microscopy revealed increased podocyte effacement and ER stress including evidence of dilated cisternae, damaged mitochondria, and abnormal autophagy in the ANLNR426C/R246C mice. Additionally, cultured primary ANLNR426C/R246C podocytes displayed increased apoptosis compared to ANLN+/+ podocytes (p=0.049).
Conclusion
ANLNR426C/R246C mice display increased susceptibility to glomerular injury when compared to ANLN+/+ littermates. Additionally, ANLNR426C/R246C mouse podocytes displayed similar ER stress and apoptotic phenotypes to cultured ANLNR431C human podocytes, lending further credibility to these results. Further evaluation of this mouse line in a genetic background that is more kidney susceptible to kidney disease should provide a necessary model to evaluate potential therapeutic compounds that have successfully rescued apoptotic phenotypes in cultured ANLNR431C human podocytes.
Funding
- NIDDK Support