Abstract: SA-PO425
Tunneling Nanotubes Shuttle Lysosomes with Low-Level α-Galactosidase A from Non-Fabry to Fabry Podocytes In Vitro
Session Information
- Genetic Diseases of the Kidney - III
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1002 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Dastvan, Frank, University of Washington, Seattle, Washington, United States
- Najafian, Behzad, University of Washington, Seattle, Washington, United States
Background
Fabry disease is an X-linked disease; however, females can suffer from serious complications. Biopsy studies are suggestive of no efficient cross-correction between non-Fabry and Fabry podocytes. We aimed to examine if there is any level of cross-correction between these cells in-vitro.
Methods
A conditionally immortalized human podocyte cell line with complete knock out of GLA (GLA-ko) using CRISPR-Cas9 was developed. Wild type (WT) podocytes were co-cultured with GLA-ko cells for a week, after which, GLA-ko and WT cells were separated by FACS sorting based on GFP expression in GLA-ko cells. Purity of sorted cells was confirmed by genotyping. Sorted co-cultured (CC) WT and GLA-ko, no co-culture (no-CC) WT and GLA-ko cells and culture media were tested for GLA-mRNA (qPCR), α-Gal-A [western blot (WB) and immunofluorescence (IF)], and α-Gal-A activity.
Results
GLA-mRNA in CC-GLA-ko cells was 2.6 fold less than WT podocytes, but 2.7 fold greater than no-CC-GLA-ko podocytes. No GLA-mRNA was detected in culture media. By WB, while no α-Gal-A protein was detected in no-CC-GLA-ko cells, this was present in CC-GLA-ko cells, albeit being 6 fold less than in WT podocytes, confirmed by IF for α-Gal-A. There was very scant α-Gal-A in media from WT and co-cultured podocytes by WB (31 fold and 15 fold less than intracellular WT, respectively). While there was almost no α-Gal-A activity in no-CC-GLA-ko podocytes, this was present in CC-GLA-ko cells, albeit being 6 fold less than in WT podocytes. There was no detectable α-Gal-A mRNA or enzyme activity in the media. A survival assay showed similarly reduced cellular proliferation in both CC-GLA-ko and no-CC-GLA-ko podocytes compared to CC-WT or no-CC-WT podocytes. IF staining showed tunneling nanotubes (TNTs) containing lysosomes and α-Gal-A running between the cells.
Conclusion
Our data suggest that there is small transfer of GLA-mRNA and α-Gal-A protein from non-Fabry to Fabry podocytes through TNTs. This low level cross-correction led to small increase in α-Gal-A activity in Fabry podocytes but was not enough to improve survival of these cells. It will be important to demonstrate if this phenomenon exists in-vivo and between other cells. These studies may lead to novel treatment options for females with Fabry disease.
Funding
- Commercial Support –