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Abstract: TH-PO762

Uncovering the Role of Cyclin G-Associated Kinase (GAK) in Regulating the Podocyte Cytoskeleton

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Bunda, Patricia, Yale University, New Haven, Connecticut, United States
  • Tian, Xuefei, Yale University, New Haven, Connecticut, United States
  • Inoue, Kazunori, Yale University, New Haven, Connecticut, United States
  • Ishibe, Shuta, Yale University, New Haven, Connecticut, United States
Background

Podocytes are terminally differentiated epithelial cells that are integral components of the glomerular filtration barrier (GFB). These cells have specialized interdigitating structures called foot processes, characterized by an extensive network of actin filaments. Damage to podocytes leads to cytoskeletal reorganization and morphological changes, which ultimately result to foot process effacement and disruption of the GFB. In 2020, our group previously identified subsets of chronic kidney disease (CKD) patients with significantly reduced expression of cyclin G associated kinase (GAK).

Methods

We subsequently generated a podocyte specific Gak knockout mouse (Gakfl/fl Pod-Cre-Dtrfl) mice, which developed foot processes effacement, progressive proteinuria, and profound glomerulosclerosis. To further interrogate the role of GAK in podocytes, we utilized a mouse model, which enables transgenic expression of a truncated GAK protein consisting only of the 62-kDa C-terminus end (Gak-C62+/−). We then generated the C62/Gak KO mouse model by crossing the Gak-C62+/− mouse to our podocyte specific Gak KO mouse.

Results

Interestingly, our preliminary data showed that the C62/Gak KO mice rescued the proteinuria and glomerulosclerosis phenotypes, and podocyte foot processes were also maintained, suggesting a novel role of the C-terminal domains of GAK in cytoskeletal regulation in podocytes. Furthermore, in vitro analysis of cell morphology indicates disruption of cytoskeletal structure of Gak KO podocytes compared to wildtype controls. Gak KO podocytes form long spindles as opposed to the stellate shaped wildtype podocytes. This morphological change in vitro was again rescued in podocytes isolated from the C62/Gak KO mouse. To elucidate the mechanism of rescue, we also performed a co-immunoprecipitation combined with shotgun proteomics experiment to identify potential binding partners of the C-terminus end of GAK. Remarkably, our results suggest a novel interaction between GAK and lymphocyte-specific protein 1 (LSP1), a known actin-binding partner, which was the top hit.

Conclusion

Together, these results further support the integral role of GAK, specifically its 62-kDa C-terminus in regulating actin dynamics in podocytes and may provide a novel target for treating CKD that specifically addresses podocyte dysfunction.

Funding

  • NIDDK Support