Kidney Week

Abstract: FR-OR061

Podocyte Cytoskeletal Stabilization Through RhoA-Cofilin Signaling by Suppressing PAI-1

Session Information

• Pathology: Novel Mechanisms and Modalities
  November 07, 2025 | Location: Room 371A, Convention Center
  Abstract Time: 05:30 PM - 05:40 PM

Category: Pathology and Lab Medicine

• 1800 Pathology and Lab Medicine

Authors

• Zhong, Jianyong, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Jianyong Zhong, MD, PhD

• Singhi, Dylan K, University of South Carolina School of Medicine Greenville, Greenville, South Carolina, United States

Dylan K Singhi

• Matsusaka, Taiji, Tokai Daigaku, Shibuya, Tokyo, Japan

Taiji Matsusaka, MD, PhD

• Yang, Haichun, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Haichun Yang, MD, PhD

• Fogo, Agnes B., Vanderbilt University Medical Center, Nashville, Tennessee, United States

Agnes B. Fogo, MD

Background

Plasminogen activator inhibitor-1 (PAI-1) is upregulated in various fibrotic kidney diseases. Our previous studies demonstrated that systemic PAI-1 knockout (KO) mitigates glomerulosclerosis and decreases proteinuria by protecting podocytes in chronic kidney disease models. In this study, we investigated whether PAI-1 has direct podocyte effects and explored the underlying mechanisms.

Methods

We generated inducible podocyte-specific PAI-1 knockdown mice (PAI-1 KD, PAI-1^floxed/podocin Cre⁺) and wild type control (WT, PAI-1^floxed/podocin Cre^-). We crossed them with Nphs1-hCD25 mice (Nep25), which express human CD25 specifically in podocytes, and develop primary podocyte injury upon injection with immunotoxin (LMB2). These mice were then assessed at 3 wks after LMB2 for podocyte injury and urine albumin/creatinine ratio (ACR). For in vitro studies, primary podocytes were isolated from WT and PAI-1 KO mice and exposed to puromycin to induce injury. RNA sequencing was performed. RhoA activity was measured using G-LISA assay and phosphorylated cofilin (p-cofilin) levels were quantified by Western Blot. Actin cytoskeleton organization was evaluated by immunofluorescent staining.

Results

PAI-1 KD/Nep25 vs Nep25 mice injected with LMB2 showed reduced ACR, less glomerulosclerosis and glomerular collagen IV deposition, with increased WT-1 positive cell density, a marker of mature podocytes. RNA sequencing of cultured PAI-1 KO vs WT podocytes after injury revealed upregulation of 3,387 genes and downregulation of 1,617 genes in the PAI-1 KO. Gene Ontology enrichment and KEGG pathway analyses revealed significant changes in genes involved in cytoskeletal organization. The activity of RhoA, a key regulator of the actin dynamics, was elevated in injured PAI-1 KO compared to WT podocytes. p-cofilin, a downstream target of RhoA that inhibits actin polymerization, was less in PAI-1 KO podocytes, thereby promoting the formation of actin filaments. WT podocytes displayed cell retraction and F-actin disorganization in response to puromycin-induced injury, while PAI-1 KO podocytes maintained intracellular F-actin but had comparable cell retraction.

Conclusion

PAI-1 deficiency in podocytes enhances RhoA activity, reduces p-cofilin levels and stabilizes the actin cytoskeleton, thereby improving podocyte structural integrity and protecting against injury.

Funding

• NIDDK Support

Digital Object Identifier (DOI)

• doi: 10.1681/ASN.2025gmgvc0xs