Kidney Week

Abstract: FR-PO761

Endosomal Recycling and Proteasomal Degradation of R168H Variant Podocin Contributes to Nephrotic Syndrome Pathophysiology

Session Information

• Glomerular Diseases: Mechanisms and Podocyte Biology
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

• Dorison, Aude, Murdoch Children's Research Institute, Parkville, Victoria, Australia

Aude Dorison, PhD

• Santos, Inês Paz, Novo Nordisk Foundation Center for Stem Cell Medicine - reNEW Copenhagen, Copenhagen, Denmark

Inês Paz Santos, MSc

• Ghobrial, Irene, Murdoch Children's Research Institute, Parkville, Victoria, Australia

Irene Ghobrial

• Lam, Rachel, Murdoch Children's Research Institute, Parkville, Victoria, Australia

Rachel Lam

• Stuber, Katrin, NNF Center for Protein Research, University of Copenhagen, Copenhagen, Denmark

Katrin Stuber

• Danne, Jillian Clare, Monash University, Melbourne, Victoria, Australia

Jillian Clare Danne, PhD

• See, Michael M., Murdoch Children's Research Institute, Parkville, Victoria, Australia

Michael M. See

• Ramm, Georg, Monash University, Melbourne, Victoria, Australia

Georg Ramm, PhD, DrPH

• Rossello, Fernando Jaime, Murdoch Children's Research Institute, Parkville, Victoria, Australia

Fernando Jaime Rossello, PhD

• Ramialison, Mirana, Murdoch Children's Research Institute, Parkville, Victoria, Australia

Mirana Ramialison

• Emdal, Kristina B., NNF Center for Protein Research, University of Copenhagen, Copenhagen, Denmark

Kristina B. Emdal, MSc, PhD

• Little, Melissa H., Murdoch Children's Research Institute, Parkville, Victoria, Australia

Melissa H. Little, PhD

Background

Nephrotic syndrome (NS) arising from NPHS2 variants is most common in children over 3 months of age. Using stem-cell derived kidney organoid podocytes, we previously reported distinct mistrafficking of specific missense NPHS2 variants, with a distinct Golgi accumulation of the p.R168H variant podocin. While a novel mislocalisation for this variant, the pathophysiology of this mistrafficking remained unclear.

Methods

Induced human pluripotent stem cells generated from a homozygous (HOM) NPHS2 p.R168H patient, healthy heterozygous (HET) parent and healthy wild type (WT) individual were differentiated into kidney organoids. Organoid glomeruli isolated from each genotype were analysed by transcriptomics and proteomics followed by gene set enrichment analyses. Immunostaining and immunogold electron microscopy (EM) were performed to validate omics data and study PODOCIN subcellular localisation in podocytes.

Results

Glomerular proteomics revealed significant differences in protein prevalence and ubiquitination between HOM and healthy podocytes. Reduced diglycine-modified lysine residues suggested preferential inhibition of YAP1 ubiquitination in HOM podocytes. This was supported by a trend of increased nuclear translocation for this transcription coregulator. A combined analysis of proteomics and transcriptomics revealed reduced ER-to-Golgi trafficking (decreased KDELR1/2, SURF4 and PDCD6) but increased recycling endosome activity (elevated RAB25) in HOM podocytes, suggesting a return from the membrane to the Golgi of the variant protein. This was confirmed using immunogold EM which detected p.R168H at the membrane, in multivesicular bodies, endosomes and Golgi while the WT protein was mainly located at the membrane. Finally, while lysosomal trafficking was reduced, p.R168H protein levels were elevated upon treatment with a proteasomal inhibitor, Bortezomib.

Conclusion

Our data indicate that p.R168H variant podocin can reach the slit-diaphragm but is then recycled back to the Golgi and/or targeted for degradation by the proteasome. This variant-specific pathophysiology will assist in the design of treatments for this form of genetic NS.

Funding

• Government Support – Non-U.S.