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Abstract: PO1663

The CLVS1 H310Y Variant Associated with Steroid-Responsive Nephrotic Syndrome Affects Podocyte Function and Glomerular Filtration Barrier Integrity

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Lane, Brandon M., Duke University Department of Pediatrics, Durham, North Carolina, United States
  • Chryst-Stangl, Megan, Duke Molecular Physiology Institute, Duke University, Durham, North Carolina, United States
  • Shalaby, Mohamed Ahmed, King Abdulaziz University Hospital, Jeddah, Saudi Arabia
  • El desoky, Sherif Mohamed, King Abdulaziz University Hospital, Jeddah, Saudi Arabia
  • Wu, Guanghong, Duke Molecular Physiology Institute, Duke University, Durham, North Carolina, United States
  • Vancini, Ricardo, Duke University School of Medicine, Durham, North Carolina, United States
  • Howell, David Noble, Duke University Department of Pathology, Durham, North Carolina, United States
  • Hall, Gentzon, Duke University Hospital, Durham, North Carolina, United States
  • Kari, Jameela Abdulaziz, King Abdulaziz University Hospital, Jeddah, Saudi Arabia
  • Gbadegesin, Rasheed A., Duke University Department of Pediatrics, Durham, North Carolina, United States
Background

We identified a rare homozygous variant, p.H310Y, in the gene encoding clavesin1 (CLVS1) as a novel cause of steroid sensitive nephrotic syndrome (SSNS). Knockdown of the orthologous CLVS1 gene in zebrafish resulted in edema phenotypes that could be rescued with WT CLVS1 mRNA but not the H310Y variant. CLVS1 knockout in cultured human podocytes decreased endocytosis, increased reactive oxygen species (ROS) accumulation, and increased apoptosis. These aberrant phenotypes were rescued in the presence of glucocorticoids, mimicking the steroid responsive phenotype of CLVS1 H310Y patients. Treatment with ROS inhibitors also rescued the reduced viability phenotype in CLVS1 KO podocytes.

Methods

To better understand the effects of the CLVS1 H310Y variant on podocyte homeostasis, we created human podocyte cell lines with CRISPR-Cas9 mediated heterozygous and homozygous CLVS1 H310Y knock-in (KI) mutations. We evaluated the KO and KI podocytes through automated live-cell imaging. Additionally, we further evaluated the effects of reduced CLVS1 function on podocyte function in vivo in zebrafish.

Results

Clavesin1 is specifically required for clathrin mediated endocytosis in cultured human podocytes. Additionally, homozygous H310Y KI podocytes displayed similar corticosteroid responsive phenotypes to CLVS1 KO lines, including increased apoptosis that could be rescued with ROS inhibition, while heterozygous KI lines were unaffected. Furthermore, we confirmed that the H310Y variant reduces binding to a critical antioxidant transporter, alpha tocopherol transfer protein (p=0.006), likely contributing to the ROS phenotypes. Electron microscopy analysis and quantification of excreted proteins revealed podocyte effacement and decreased glomerular filtration barrier integrity in zebrafish with knockdown of orthologous CLVS1 when compared to controls (p<0.0001).

Conclusion

Our data further demonstrates the importance of clavesin1 in the maintenance of podocyte viability and GFB integrity. It also suggests that oxidative stress regulation may be compromised in patients carrying pathogenic CLVS1 variants and highlights the potential for alternative therapies for NS patients that target ROS accumulation in podocytes.

Funding

  • Other NIH Support