Abstract: SA-PO307
Complete Lack of Synaptopodin Causes No Overt Podocyte Defects
Session Information
- Cellular Crosstalk in Glomerular Diseases - II
October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix
Authors
- Ning, Liang, Washington University, St. Louis, Missouri, United States
- Suleiman, Hani, Washington University, St. Louis, Missouri, United States
- Miner, Jeffrey H., Washington University School of Medicine, St. Louis, Missouri, United States
Background
Synaptopodin (Synpo) is an actin-associated protein found in podocyte foot processes and in dendritic spines. Many functions in regulating the actin cytoskeleton via RhoA and other pathways have been ascribed to Synpo, yet no pathogenic mutations in SYNPO have been discovered in patients. Several naturally occurring Synpo isoforms have been described (e.g., Synpo-short and -long), and a novel truncated protein (Synpo-T) was detected in the podocytes of Synpomutant mice. Synpo-T was reported to maintain some Synpo functions, thus preventing a major podocyte phenotype from emerging. To further investigate the functions of Synpo in podocytes, we attempted to knockout the majority of the Synpogene and prevent production of a protein.
Methods
We used CRISPR/Cas9 in embryos with two guide RNAs targeting near the 5’ and 3’ ends of the Synpo gene to attempt to either delete most of the gene or mutate each end to prevent protein production. DNA sequencing was used to identify specific mutations. Immunofluorescence and western blotting using antibodies to amino-terminal, internal, and carboxyl-terminal epitopes were used to assay for Synpo protein. Mutant mice were observed for up to 12 months. Urine, blood, and tissue were taken at select time points to evaluate albuminuria, BUN, histopathology, and glomerular capillary wall ultrastructure. Super-resolution imaging was used to characterize the cytoskeleton. Adriamycin injections were performed.
Results
Many mutations were discovered in 80 founder mice: small deletions and insertions that shifted the reading frame; two deletions of 8 kb between the gRNA target sites; a 6 kb inversion; and a 3’ deletion of 7 bp that produced a truncated protein. Several of these were made homozygous or compound heterozygous. There were no significant differences in body weight between the mutants and controls. Urinary protein levels were normal up to 12 months regardless of the Synpo mutation(s) present. There were no obvious histological or ultrastructural abnormalities in 36-week-old mutant mice, and the actin cytoskeleton showed no obvious defects other than the absence of Synpo. However, Synpo mutant podocytes had worse injury from ADR.
Conclusion
Synaptopodin is dispensable for the development and maintenance of podocytes and for function of the glomerular filtration barrier. This is consistent with the lack of SYNPO mutations found in proteinuric patients.
Funding
- NIDDK Support