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Abstract: SA-OR27

RCAN1 I162T Variant Disrupts Calcineurin Regulation and Reduces Viability in Patient-Derived Podocytes

Session Information

Category: Glomerular Diseases

  • 1304 Glomerular Diseases: Podocyte Biology

Authors

  • Huggins, Kinsie, Duke University, Durham, North Carolina, United States
  • Datta, Somenath, Duke University School of Medicine, Durham, North Carolina, United States
  • Soldano, Karen, Duke University School of Medicine, Durham, North Carolina, United States
  • Silas, Daniel Philip, Duke University School of Medicine, Durham, North Carolina, United States
  • Elhassan, Elhussein Aamir Elzein, Beaumont Hospital, Dublin, Ireland
  • Chryst-Stangl, Megan, Duke University School of Medicine, Durham, North Carolina, United States
  • Wu, Guanghong, Duke University School of Medicine, Durham, North Carolina, United States
  • Olabisi, Opeyemi A., Duke University School of Medicine, Durham, North Carolina, United States
  • Conlon, Peter J., Beaumont Hospital, Dublin, Ireland
  • Gbadegesin, Rasheed A., Duke University School of Medicine, Durham, North Carolina, United States
  • Lane, Brandon M., Duke University School of Medicine, Durham, North Carolina, United States
Background

We have previously shown that deleterious mutations in RCAN1 are a cause of familial FSGS. One of these mutations, I162T resulted in dysregulated calcineurin (CN) activity and reduced viability in HEK293 cells overexpressing the variant compared to WT expressing cells. Deficits in I162T cell viability was rescued by treatment with calcineurin inhibitors as well as inhibitors of GSK-3β. Knockdown of RCAN1 also produced similar disruptions in podocyte viability in conditionally immortalized human podocytes. However the phenotype of podocytes derived from patients with RCAN1 I162T mutation is unknown.

Methods

Using PBMCs collected from a patient carrying the RCAN1 I162T mutation as well as an unaffected family member, we generated induced pluripotent stem cells (iPSCs). After differentiating iPSCs into podocytes, the cells were examined for changes in CN regulation and viability.

Results

Differentiated podocytes from both affected and unaffected individuals displayed podocyte specific markers including Nephrin, WT1 and Synaptopodin as confirmed by both RT-PCR and immunofluorescence. Automated live-cell imaging over 72 hours using a fluorescent reporter of caspase 3 activity revealed an increase in serum starvation induced apoptosis in the podocytes carrying the mutant I162T compared to podocytes from the control unaffected family member (p<0.0001).

Conclusion

We have now confirmed that the RCAN1 I162T allele causes decreased podocyte viability due to defects in calcineurin regulation using cells derived from affected family. These patient iPSC derived podocytes set the stage for a better understanding of RCAN1-mediated regulation of CN activity and evaluation of therapeutic alternatives to calcineurin inhibitors in the treatment of FSGS.

Funding

  • NIDDK Support