Abstract: FR-PO361

STAT3 Regulates Fibrogenic Signaling in Pericytes and Activates Migration, Differentiation, and Secretion of Pro-Fibrotic Cytokines

Session Information

Category: Chronic Kidney Disease (Non-Dialysis)

  • 308 CKD: Mechanisms of Tubulointerstitial Fibrosis

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 Women's Hospital, Boston, Massachusetts, 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 plays an important role in cell proliferation, cellular pluripotency, and differentiation. Here, we investigated the pathophysiological role of STAT3 signaling in kidney fibrosis.

Methods

Stromal cell-specific STAT3 deletion was performed by breeding STAT3 floxed mice with FoxD1 Cre mice. Kidney fibrosis was induced by injecting a single dose of 300 mg/kg body weight folic acid (FA) or 5 mg/kg body weight aristolochic acid (AA). Immunostaining for STAT3 phosphorylations (Ser727 and Tyr705) was performed on mice kidneys and 10T1/2 cells. We developed two activation mutants and one-inactivation mutant of STAT3 using CRISPR-Cas9 technology in pericytes-like (10T1/2) cells. Cell migration was evaluated with Boyden chambers and wound scratch assays. Cell proliferation was measured by MTT assay. RT-PCR and western blotting were used to measure STAT3 dependent genes and luminex-based assay of cytokines (CTGF, TGF-β and IL-6) were performed to quantitate pro-fibrotic cytokines.

Results

STAT3 phosphorylation was increased in tubular epithelial cells and pericytes of 5 human subjects with chronic kidney disease. Deletion of STAT3 in pericytes protects against FA or AA-induced kidney fibrosis at 7 and 14 days post-treatment. Fibrotic markers, including fibronectin, collagen1a1 and α-smooth muscle actin (α-SMA), were reduced in STAT3 knockout mice. In vitro, CRISPR-Cas9 mediated activation of STAT3 caused increased cell migration whereas the inhibition of STAT3 was associated with decreased migration of 10T1/2 cells. Treatment of cells with TGF-β increased the production of profibrotic cytokines and differentiation of 10T1/2 cells to myofibroblasts. Pretreatment with stattic, a small molecule inhibitor of STAT3 inhibited both cytokines production and cell differentiation.

Conclusion

Inhibition of STAT3 in pericytes protects mice from kidney fibrosis and the specific inhibition of STAT3 signaling in pericytes prevents their differentiation into myofibroblasts.

Funding

  • NIDDK Support