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Abstract: TH-PO1077

The Critical Role of Rho Associated Coiled-Coil Containing Protein Kinase 2 for the Enhancement of Actomyosin Contractility in Podocytes

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Author

  • Lee, Hsiao-Hui, National Yang-Ming University, Taipei, Taiwan

Group or Team Name

  • Cytoskeleton and Cell Mechanics
Background

The highly differentiated podocytes elaborate interdigitated foot processes (FP) to cover the glomerular capillaries and form slit diaphragm (SD) for blood filtration. Therefore, podocytes adhere tightly to glomerular basement membrane (GBM) and generate RhoA/ROCK-mediated contraction force to tolerate the filtration pressure. Two ROCK isoforms, ROCK1 and ROCK2, were identified in mammal. In this study, we characterized that ROCK2 is critical for the enhancement of contractility in cultured podocytes.

Methods

The conditionally immortalized mouse podocytes were used in this study. By Western blotting and immunostaining, the ROCK signaling and cell architecture were detected. ROCK kinase activity was inhibited by the treatment of cells with Y27632 and ROCK2 gene was knockout by CRISPR/Cas9 method.

Results

After temperature switch, the expression of podocyte differentiation marker synaptopodin was increased. The formation of focal adhesions (FA), stress fibers and the SD-like protrusions labelled by ZO-1 staining were also observed. We found that ROCK2, but not ROCK1, was significantly activated in differentiated cells. The phosphorylation status of MLC and ERM, two ROCK downstream substrates, were also increased. treatment of cells with Y27632 diminished both the phosphorylation of MLC and ERM and the formation of FA and stress fibers. We then generated ROCK2 knockout podocytes by CRISPR/Cas9 and found that loss of ROCK2 decreased MLC phosphorylation, while ERM phosphorylation was not affected. In addition, ROCK2-dificiency also diminished the formation and stress fibers and cellular protrusions.

Conclusion

Our results suggest that ROCK2 is critical for the enhancement of actomyosin contractility in podocytes. The regulation of ROCK2 activation shall plays an important role in the maintenance of cell adhesion and cytoskeletal architecture for podocyte function.

Funding

  • Government Support - Non-U.S.