Abstract: SA-PO219
Podocytes from 129S1 Mouse Glomerular Outgrowths Can Be Used for Functional Studies
Session Information
- Glomerular: Cell Biology
November 04, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Glomerular
- 1003 Glomerular: Cell Biology
Authors
- Wang, Hong, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut, United States
- Lincoln, Kathleen A., Boehringer-Ingelheim Pharmaceuticals, Ridgefield, Connecticut, United States
- Bartlett, Christina S., Boehringer Ingelheim, Ridgefield, Connecticut, United States
- Kuo, Jay, Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Connecticut, United States
- Pullen, Steven S., Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut, United States
Background
Podocytes are specialized, terminally differentiated epithelial cells which are essential for integrity of the glomerular filtration barrier. Efforts to develop podocyte-directed therapies have been hampered by lack of in vitro systems for studying podocyte functions. Immortalized podocyte cell lines fail to represent native gene expression profiles, while the anatomic location of podocytes makes it difficult to study cell behaviors ex vivo. The 129S mouse strain is susceptible to and commonly used for models of renal disease, while currently primary mouse podocyte culture has been limited to use of the resistant C57BL/J strain. Therefore, we sought to isolate and characterize podocytes from glomerular outgrowths from 129S1 mice, a strain for which podocyte glomerular outgrowths have not been evaluated.
Methods
Glomeruli were isolated from 129S1/Svlm mice using Dynabead perfusion and magnetic isolation. We modified our protocol to include a brief collagenase incubation and MACS cell dissociation step to reduce handling time and increase viability. Purified glomeruli were resuspended in culture media and seeded on collagen IV coated plates. Intact glomeruli were removed after 4 days in culture. After 6 days, cells were treated with puromycin aminonucleoside (PAN) for 24 hours. Podocyte markers were measured using Taqman gene expression, and F-actin was visualized using fluorescent phalloidin.
Results
Expression of podocyte specific markers (nephrin, podocin, podocalyxin, synaptopodin, WT1, Glepp-1) was observed at 6 days of culture from 129S1 glomerular outgrowths. Nephrin gene expression was lost by 10 days of culture, although expression of other podocyte markers persisted. PAN treatment dose dependently up-regulated apoptotic genes, Caspase-3 and BAD, while podocyte markers and integrin gene expression were significantly reduced. Phallodin staining showed highly organized actin fibers present within the cytoplasm of cultured cells and was reduced by PAN treatment.
Conclusion
We successfully cultured primary mouse podocytes from the 129S1 mouse strain, modified the standard protocol to reduce glomerular isolation time, and identified the useful window of utility. This method will be valuable for rapid evaluation of genetic mouse models of podocytopathy and development of assays for podocyte functional assessment.