Abstract: FR-PO348
Expression of an Anti-Fibrotic Molecule Smad Anchor for Receptor Activation (SARA) Is Differentially Regulated in Epithelial Cells and Fibroblasts at a Translational Level
Session Information
- CKD: Mechanisms - II
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2103 CKD (Non-Dialysis): Mechanisms
Authors
- Dalal, Vidhi, Lurie Children's Hospital of Chicago , Chicago, Illinois, United States
- Liang, Xiaoyan, Northwestern University, Chicago, Illinois, United States
- Hao, Xingyue Delan, Northwestern University, Chicago, Illinois, United States
- Schnaper, H. William, Northwestern University, Chicago, Illinois, United States
- Hayashida, Tomoko, Northwestern University, Chicago, Illinois, United States
Background
We reported last ASN meeting that overexpression of SARA specifically in pericytes prevents them from transdifferentiating into fibroblasts, hence mitigates fibrotic changes in a mouse model of interstitial fibrosis. These findings suggest that SARA is a key molecule that regulates cellular phenotype. Here, we aimed to explore the mechanism by which SARA levels are regulated.
Methods
SARA protein and mRNA expression levels were evaluated in cultured epithelial cells and fibroblasts derived from rat kidney (NRK52E and 49F, respectively), with or without transfection of a plasmid expressing human SARA cDNA or HA-tagged focal adhesion kinase (FAK). RNA from NRK52E and 49F was subjected to RNA sequencing.
Results
SARA protein was abundantly expressed in NRK52E, while it was barely detected in NRK49F. In contrast, SARA mRNA levels were similar in NRK52E and 49F. When a plasmid expressing human SARA driven by a constitutive promoter was transfected, SARA protein overexpression was apparent in NRK52E cells, but not in 49F, while HA-FAK protein expression used as a control was equivalent both in NRK52E and 49F, indicating that the difference in 52E and 49F are not due to transfection efficiency. Indeed, human SARA RNA driven by SARA overexpression plasmid was significantly and equally increased in both NRK52E and 49F. RNA sequencing revealed 107,000 genes expressed both in NRK52E and 49F, and 922 and 754 genes were unique to NRK52E and 49F, respectively. KEGG enrichment analyses revealed that genes associated with metabolic pathways were most commonly differentially regulated (N=630), followed by those associated with PI3K-Akt signaling pathways (N=197) and endocytosis (N=176). Most significantly enriched genes were found in adherens junction related genes, and Hippo signaling and HIF-1 signaling pathway-related genes.
Conclusion
These results suggest that, in fibroblasts, a mechanism exists that keeps SARA levels low at a translational level. Some of the differentially expressed genes revealed by RNA sequencing in epithelial cells and fibroblasts are likely to be involved this regulation and could be a novel therapeutic target.
Funding
- NIDDK Support