Abstract: FR-PO1212
Characterizing New Negative Regulators of Tubular GEF-H1 Signaling: Possible Role in Kidney Fibrosis
Session Information
- CKD: Mechanisms, AKI, and Beyond - 2
November 07, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Szaszi, Katalin, St Michael's Hospital Keenan Research Centre for Biomedical Science, Toronto, Ontario, Canada
- Wu, Brian Rai-Ling, St Michael's Hospital Keenan Research Centre for Biomedical Science, Toronto, Ontario, Canada
- Sri Theivakadadcham, Veroni S, St Michael's Hospital Keenan Research Centre for Biomedical Science, Toronto, Ontario, Canada
- Dan, Qinghong, St Michael's Hospital Keenan Research Centre for Biomedical Science, Toronto, Ontario, Canada
- Venugopal, Shruthi, St Michael's Hospital Keenan Research Centre for Biomedical Science, Toronto, Ontario, Canada
- Kapus, Andras, St Michael's Hospital Keenan Research Centre for Biomedical Science, Toronto, Ontario, Canada
Background
Kidney fibrosis is attributed to chronic injury, inflammation and maladaptive repair caused by kidney diseases of various etiologies. Tubular RhoA proteins, activated by cytokines, regulate release of fibrogenic mediators, including Connective Tissue Growth Factor (CTGF/CCN2). These in turn stimulate mesenchymal cells to produce extracellular matrix proteins. This epithelial-mesenchymal crosstalk is crucial for fibrogenesis. The guanine nucleotide exchange factor GEF-H1 (ArhGEF2), activated and upregulated by inflammatory and pro-fibrotic input, is an activator of tubular fibrogenic RhoA signaling. The dynamic activation-inactivation cycle of GEF-H1, and its fibrosis-related dysregulation however remain poorly understood. This study aimed at identifying new druggable regulators of fibrogenic GEF-H1/RhoA signaling and tubular cytokine production.
Methods
LLC-PK1 and hTERT-immortalized human renal proximal tubule cells were used. Proteins coimmunoprecipitating with GEF-H1 were identified using mass spectrometry and verified by western blotting. Expression of key proteins were altered by siRNA-mediated silencing or overexpression. RhoA and GEF-H1 activity were assessed by affinity precipitation assays. Fibrosis-related genes were measured using RT2 Profiler™ PCR Array (Qiagen). Protein and mRNA expression changes were explored in a mouse kidney fibrosis model.
Results
GEF-H1 associated with subunits of protein phosphatase 6 (PP6) and with Myotonic Dystrophy Kinase-related Cdc42-binding kinase (MRCK)α. Silencing of either the PP6 catalytic domain (PP6C) or MRCKα stimulated GEF-H1 activity and promoted GEF-H1-dependent RhoA activation. We identified several genes upregulated by depletion of PP6C or MRCKα. Notably, depletion of PP6C or MRCKα activated the RhoA effector Myocardin-related Transcription Factor (MRTF), and elevated MRTF-dependent genes, including CTGF/CCN2 and α-smooth muscle actin. Finally, expression of PP6C was decreased in a mouse kidney fibrosis model and in tubular cells stimulated by TGFβ1.
Conclusion
We identified PP6 and MRCKα as new suppressors of GEF-H1/RhoA signaling and tubular fibrotic changes. Cytokine-induced downregulation of these regulators may contribute to epithelial-mesenchymal crosstalk and could represent new drug targets for kidney fibrosis.
Funding
- Government Support – Non-U.S.