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Abstract: TH-PO990

Deletion of Ste20-Like Kinase SLK Induces Cytoskeletal Changes in Podocytes

Session Information

Category: Pathology and Lab Medicine

  • 1501 Pathology and Lab Medicine: Basic

Authors

  • Woychyshyn, Boyan, McGill University , Montreal, Quebec, Canada
  • Papillon, Joan, McGill University , Montreal, Quebec, Canada
  • Guillemette, Julie, McGill University , Montreal, Quebec, Canada
  • Cybulsky, Andrey V., McGill University , Montreal, Quebec, Canada
Background

Glomerular epithelial cells (GECs; podocytes) are essential in maintaining glomerular permselectivity. Their function is dependent on an intact actin cytoskeleton. Regulation of the actin cytoskeleton, in part, relies on the assembly of a phospho-ezrin-NHERF2-podocalyxin complex at the apical surface of the podocyte. SLK is a serine/threonine kinase ubiquitously expressed in mammalian tissues, including GECs. SLK was reported to be involved in the arrangement of the actin cytoskeleton and in mediating cell cycle progression. Deletion of SLK in podocytes in mice leads to proteinuria and disruption of podocyte architecture as mice age. The aim of this study was to determine if SLK phosphorylates ezrin in podocytes and is critical in maintaining cytoskeletal integrity.

Methods

We generated a SLK knockout (KO) GEC line by transducing immortalized GECs isolated from mice carrying a floxed SLK allele with tamoxifen-inducible Cre recombinase. We also depleted SLK in GECs using siRNAs. We studied the effect of SLK deletion on phosphorylated ezrin, podocalyxin and F-actin using immunoblotting and immunofluorescence microscopy.

Results

In GECs, both KO of SLK using Cre recombinase and depletion of SLK with siRNAs reduced SLK protein expression and pT183 phosphorylation, which reflects catalytic activity (KO being more effective than siRNA). SLK KO did not affect the rate of proliferation of GECs. Both SLK KO and depletion markedly reduced activation-specific phosphorylation of ezrin, while total ezrin protein levels remained unchanged. Expression of podocalyxin, a protein essential to foot process integrity, was significantly reduced in the SLK KO cells. Levels of F-actin were markedly reduced in SLK KO cells and stress fiber arrangements were altered; however, total actin was unaffected. Treatment of GECs with erlotinib, a chemical inhibitor of SLK, showed effects on F-actin similar to KO of SLK.

Conclusion

SLK is not essential for cell cycle progression in GECs. SLK phosphorylates ezrin, and decreased ezrin phosphorylation due to deletion of SLK leads to reduction in podocalyxin and disruption of the actin cytoskeleton. The result suggests that SLK is important in the maintenance of the ezrin-NHERF2-podocalyxin complex and foot process integrity.

Funding

  • Government Support - Non-U.S.