Abstract: FR-PO1021
Ablation of PFN1 Expression Causes Proteinuric Disease
Session Information
- Genetic Diseases of the Kidneys: Non-Cystic - II
October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidney
- 1002 Genetic Diseases of the Kidney: Non-Cystic
Authors
- Pedigo, Christopher E., Yale University, New Haven, Connecticut, United States
- Inoue, Kazunori, Yale University, New Haven, Connecticut, United States
- Tian, Xuefei, Yale University, New Haven, Connecticut, United States
- Cross, Elizabeth, Yale University, New Haven, Connecticut, United States
- Wang, Ying, Yale University, New Haven, Connecticut, United States
- Ishibe, Shuta, Yale University, New Haven, Connecticut, United States
- Li, Wei, Yale University, New Haven, Connecticut, United States
Background
Understanding the pathobiology of glomerular diseases is paramount as it accounts 80% of end stage kidney disease cases in the US. Recent reports have identified human mutations in INF2 to cause proteinuric disease leading to loss of F-actin stress fiber formation in podocytes. Our study focused on an INF2 interactor Profilin 1(PFN1), a protein involved in the elongation of F-actin fibers. Despite the discovery of reduced PFN1 expression in microdissected Diabetic Kidney Disease(DKD) patient glomeruli, the function of PFN1 in podocytes is unknown. To investigate the role of Pfn1 in podocytes we studied diabetic mice, generated podocyte specific Pfn1 knockout mice and used CRISPR-Cas9 to knockout PFN1 in cultured human podocytes.
Methods
Glomeruli from the DKD model, db/db, were isolated using dynabeads. Analysis for RNA and protein were performed. Podocyte specific Pfn1 knockout mice were generated and phenotyped. Purified primary podocytes were isolated from Pfn1KO mice by breeding to the Terminator mouse. CRISPR-Cas9 mediated PFN1 knockout (PFN1KO) cultured human podocytes were generated and analyzed.
Results
Pfn1 expression is reduced in glomeruli from db/db mice compared to controls. To understand the role PFN1 in podocytes, podocyte specific Pfn1 knockout(Pfn1KO) mice were generated. Pfn1KO mice demonstrated progressive albuminuria starting at 3 weeks(p<0.05) and severe kidney failure (p<0.05) leading to morbidity and mortality by (p<0.05) by 9 weeks of age. Histological analysis revealed increased mesangial expansion (PAS staining, p<0.05) and progressive interstitial fibrosis/proteinaceous casts (Trichrome Staining, p<0.05) by 6 weeks of age. Ultrastructural analysis revealed podocyte foot process effacement and vacuole-like structures which was recapitulated by similar structures in PFN1KO cultured human podocytes. Primary mouse Pfn1KO podocytes and PFN1KO cultured human podocytes revealed a significant reduction in ability to spread and migrate (p<0.05).
Conclusion
Our data suggest that loss of podocyte Pfn1 expression is sufficient to cause podocyte injury, identifying PFN1 as an essential regulator of podocyte function and maintenance. These data support further identification of PFN1 regulatory targets in podocytopathies.
Funding
- NIDDK Support