Abstract: TH-PO631
Generation and Validation of a Novel Primary Renal Interstitial Cell Line
Session Information
- Development, Stem Cells, Regenerative Medicine - I
October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 501 Development, Stem Cells, and Regenerative Medicine: Basic
Authors
- Mccarthy, Sarah, Maine Medical Center Research Institute, Scarborough, Maine, United States
- Oxburgh, Leif, Maine Medical Center Research Institute, Scarborough, Maine, United States
Background
The renal interstitium is indispensable for proper nephrogenesis during mammalian kidney development. Interstitial cells also play a central role in the adult kidney fibrotic response. Despite the clinical significance, limited reagents are available to examine the signaling mechanisms that govern interstitial cell biology. To this end, we have generated an immortalized primary renal interstitial cell (PRIC) line.
Canonical WNT signaling drives cellular proliferation in a number of developmental and disease contexts. Furthermore, in vivo deletion of Wnt7b from the collecting duct or β-catenin from interstitial cell precursors leads to medullary hypoplasia, suggesting a role for paracrine WNT signaling on interstitial cell maintenance. Biochemical analysis with our validated PRIC line confirmed that WNT drives renal interstitial cell proliferation.
Methods
A novel isolation method was utilized to purify PRICs from postnatal mice. Cells were then transduced with a mCherry-tagged SV40T temperature sensitive lentivirus construct to produce a heterogeneous population termed “bulk” SV40T PRICs. Clonal lines were isolated and expanded from single cells and screened for expression of postnatal interstitial zone markers. The transfection efficiency of isolated clones was evaluated with a GFP-expressing construct. The affects of treatment with the WNT agonist CHIR and the TCF/LEF1 inhibitor Fh535 on proliferation was measured by EdU labeling and ClickIt chemistry.
Results
As expected, “bulk” SV40T PRICs showed expression of SV40T at 33°C which was lost when cultured at 37°C. Clone 3-1 was selected for further characterization based on its transcriptional profile and expression of the common interstitial markers Pdgfrβ, α-SMA, Meis1, fibronectin and vimentin. The transfection efficiency of clone 3-1 was determined to be approximately 40%. Treatment of clone 3-1 with CHIR produced a time-dependent increase in the feedback pathway reporters Axin2 and Lef1. In addition, clone 3-1 exhibited a dose-dependent increase in proliferation in response to CHIR. Finally, co-treatment with Fh535 abrogated the mitotic response observed with CHIR alone.
Conclusion
These experiments confirm that clone 3-1 is a viable in vitro model to study interstitial cell biology and that canonical WNT signaling through TCF/LEF1 drives renal interstitial cell proliferation.
Funding
- NIDDK Support