Abstract: SA-PO374
Sorting Nexin 9 Facilitates Podocin Endocytosis in the Injured Podocyte
Session Information
- Glomerular Diseases: Immunology and Inflammation - III
October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Author
- Sasaki, Yu, Juntendo University, Tokyo, Japan
Background
The irreversibility of glomerulosclerotic changes depends on the degree of podocyte injury. We have previously demonstrated the endocytic translocation of podocin to the subcellular area in severely injured podocytes and found that this process is the primary disease trigger. We identified the protein sorting nexin 9 (SNX9) as a novel facilitator of podocin endocytosis in a yeast two-hybrid analysis. SNX9 is involved in clathrin-mediated endocytosis, actin rearrangement and vesicle transport regulation.
Methods
We conducted co-immunoprecipitation assays to confirm the interaction between podocin and SNX9 using lysates of FLAG-podocinand GFP-SNX9-overexpressing cells. To map the podocin binding site(s) in SNX9, we tested the abilities of various truncated GFP-SNX9 constructs to co-precipitate with FLAG-podocin. To further demonstrate that podocin can directly bind to SNX9, we performed GST pull-down assays with purified recombinant proteins. To detect SNX9 expression in kidney samples, we subjected normal and adriamycin (ADR)-injected mice to immunofluorescence staining. To evaluate SNX9 expression in vitro, we performed immunofluorescence staining on non-treated and ADR-treated cultured human podocytes. To assess SNX9 expression in human kidney glomeruli, we subjected human kidney biopsy specimens to immunofluorescence staining.
Results
Our results revealed and confirmed that SNX9 interacts with podocin exclusively through the Bin–Amphiphysin–Rvs (BAR) domain of SNX9. Immunofluorescence staining revealed the expression of SNX9 in response to podocyte adriamycin-induced injury both in vitro and in vivo. Finally, an analysis of human glomerular disease biopsy samples demonstrated strong SNX9 expression and co-localization with podocin in samples representative of severe podocyte injury, such as IgA nephropathy with poor prognosis, membranous nephropathy and focal segmental glomerulosclerosis.
Conclusion
We identified SNX9 as a facilitator of podocin endocytosis in severe podocyte injury and demonstrated the expression of SNX9 in the podocytes of both nephropathy model mice and human patients with irreversible glomerular disease.
Funding
- Government Support - Non-U.S.