Kidney Week

Abstract: TH-PO948

A Novel Conditionally Immortalized Kidney Pericyte Cell Line to Model Myofibroblast Transition

Session Information

• Molecular Mechanisms of CKD - I
  October 25, 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

• Humphreys, Benjamin D., Washington University School of Medicine, Clayton, Missouri, United States

Benjamin D. Humphreys,

• Donnelly, Erinn L., Washington University School of Medicine, Clayton, Missouri, United States

Erinn L. Donnelly,

• Chang Panesso, Monica, Washington University in St. Louis, St Louis, Missouri, United States

Monica Chang Panesso,

• O hAinmhire, Eoghainín, Washington University, Lake St. Louis, Missouri, United States

Eoghainín O hAinmhire,

• Machado, Flavia G., Washington University in St Louis, School of Medicine, St Louis, Missouri, United States

Flavia G. Machado,

Background

Gli1-positive resident pericytes and perivascular fibroblasts are the predominant source of kidney myofibroblasts in kidney fibrosis but the study of pericyte to myofibroblast transition is hampered by the absence of appropriate cell culture models.

Methods

We crossed bigenic Gli1-CreER^t2; R26tdTomato mice with the ‘immorto-mouse,’ which expresses a temperature sensitive SV40 gene, and with the ‘terminator’ mouse, which expresses the diphtheria toxin receptor except after Cre-mediated recombination. After tamoxifen administration, we isolated tdTomato-positive Gli1 cells from kidney, and elimated all non-recombined cells by administration of diphtheria toxin. We characterized these cells ability to differentiate into myofibroblasts using a variety of methods, including scRNA-seq.

Results

Kidney Pericyte-Gli1 (KPG) cells maintain expression of appropriate pericyte cell markers, respond to hedgehog pathway activation and display robust myofibroblast differentiation upon treatment with TGFb. In support of their pericyte identity, co-culture of KPG with endothelium stabilizes capillary formation. Single cell RNA-sequencing analysis (5,000 cells) during the myofibroblast differentiation timecourse identified autocrine ligand-receptor upregulation including the connective tissue growth factor, platelet-derived growth factor and transforming growth factor beta-3 pathways. Strong upregulation of genes in the focal adhesion pathway led us to test the serum response factor inhibitor CCG-203971 which potently inhibited TGFb-induced pericyte to myofibroblast transition. Finally, our single cell analysis identified the unexpected upregulation of nerve growth factor (NGF) in KPG cells treated with TGFb. We confirmed strong NGF upregulation in two mouse kidney fibrosis models, and identified expression of the NGF receptor TrkA in VSMC cells in vivo, suggesting a novel role for perivascular fibroblast-derived NGF signaling to VSMC during kidney fibrosis.

Conclusion

KPG cells accurately model pericyte to myofibroblast transition, and we used them to identify a novel inhibitor of this process and the unexpected upregulation of the NGF pathway during renal fibrosis.

Funding

• NIDDK Support