Abstract: SA-PO811
STAT3 Deletion from Stromal Cells Protects Mice from Folic Acid or Aristolochic Acid-Induced Kidney Fibrosis by Limiting Differentiation and Migration of Pericytes
Session Information
- Molecular Mechanisms of CKD - III
October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 1903 CKD (Non-Dialysis): Mechanisms
Authors
- Ajay, Amrendra Kumar, Brigham and Women's Hospital, Boston, Massachusetts, United States
- Vig, Shruti, Brigham and women''s hospital, Boston, Massachusetts, United States
- Akinfolarin, Akinwande A., Brigham And Womens Hospital, Mckinney, Texas, United States
- Sabbisetti, Venkata, Brigham and Women's Hospital , Boston, Massachusetts, United States
- Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States
Background
STAT3 is a key transcription factor, which is phosphorylated in response to growth factors and cytokines. Upon phosphorylation, STAT3 regulates cell proliferation and migration. Here, we investigated the function of STAT3 signaling in the development of kidney fibrosis.
Methods
STAT3 activation was measured in 5 patient biopsy samples. Stromal cell-specific STAT3 deletion was performed by breeding STAT3 floxed mice with FoxD1 Cre mice. Kidney fibrosis was induced by administering 300 mg/kg folic acid (FA) or 5 mg/kg body weight aristolochic acid (AA). We developed STAT3 activated pericytes like 10T1/2 cells using two CRISPR based methods: i) Synergistic Activation Mediators (SAM) sgRNA, and ii) mutation of alanine 662 and asparagine 664 residues to cysteines. Additionally, using CRISPR, we mutated serine 727 to alanine of STAT3 in pericyte cell line. Cell migration was evaluated with wound scratch assays. RT-PCR, immunostaining and western blotting were performed to measure changes in STAT3-dependent genes and to quantitate pro-fibrotic cytokines.
Results
STAT3 phosphorylation was increased in tubular epithelial cells and interstitial cells of 5 human subjects with chronic kidney disease. Deletion of STAT3 from pericytes protects mice from FA or AA-induced kidney fibrosis at 7 and 14 days post-treatment. Fibrotic markers, including fibronectin, collagen1a1, and α-SMA, were reduced in STAT3 KO mice. STAT3 KO mice show similar acute injury and have less KIM-1 expression at 7 and 14 days. CRISPR-Cas9 mediated activation and inhibition studies in vitro confirmed that STAT3 is directly regulating profibrogenic signaling in pericytes. STAT3 phosphorylation increased migration and differentiation of pericytes. STAT3 is phosphorylated after treatment with TGF-β. Inhibition of TGF-β induced STAT3 phosphorylation significantly decreased the proliferation, production of profibrotic cytokines and differentiation of pericytes.
Conclusion
STAT3 leads to fibrosis by increasing proliferation, migration and differentiation of pericytes into myofibroblasts. STAT3 is a potential therapeutic target for kidney fibrosis.
Funding
- NIDDK Support