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Abstract: FR-PO943

C-Type Lectin-Like Receptor (CLEC)-2, the Ligand of Podoplanin, Facilitates Motility of Podocytes

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Tanaka, Keiko, Tokai University School of Medicine, Isehara, KANAGAWA, Japan
  • Matsusaka, Taiji, Tokai University School of Medicine, Isehara, KANAGAWA, Japan
Background

Podoplanin is strongly expressed on podocyte membrane in an evolutionally conserved manner. Podoplanin binds to phosphorylated (p)ezrin, which binds and maintains filamentous (F-) actin. The endogenous ligand of podoplanin is C-type lectin-like receptor (CLEC)-2, which is highly expressed in platelets. Normally, podocytes are sequestered from platelets, but when the GBM barrier is injured, podocytes may have access to platelets or CLEC-2. In addition, soluble CLEC-2 was reported to exist in human serum. In the present study, we aimed to investigate the impact of CLEC-2 on podocytes.

Methods

Recombinant Fc-CLEC-2, a fusion of human Fc and CLEC-2 (51-229) proteins, which was reported to bind to mouse podoplanin, was generated in HEK293 cells and purified by Protein A affinity chromatography. Primary mouse cultured podocytes were incubated with the Fc-CLEC-2 or Fc protein (each 10 ug/mL) for 1 hour. Cell morphology, F-actin (Phalloidin staining), p-ezrin (immunostaining), cell adhesion to collagen-1 coated dish, and migration (Wound scratch test) were evaluated. To assess whether Fc-CLEC-2 has an impact on reversal from injury, podocytes were pretreated with protamine sulfate (PS) followed by heparin sulfate (HS), and then the effect of Fc-CLEC-2 or Fc on cell form, F-actin and p-ezrin were evaluated.

Results

No obvious change was observed in cell form and F-actin after the stimulation with Fc-CLEC-2. However, podocytes treated with Fc-CLEC-2 showed less adhesion to collagen-1 coated plate within 1 hour than the Fc control (86.7% vs. 100%). In migration assay, podocytes with Fc-CLEC-2 showed faster movement than control (11.1 vs. 9.2 mm/24hr) and faster wound closure (18.6 vs. 21.0 hours). After incubation with PS and HS, Fc-CLEC-2 retarded recovery of F-Actin formation compared to control (54.4% vs. 77.0%). Treatment with PS and HS increased p-ezrin, which was further enhanced by Fc. In contrast, Fc-CLEC-2 reduced cytosolic p-ezrin, suggesting that CLEC-2 inhibits F-actin formation by decreasing p-ezrin which binds to podoplanin.

Conclusion

These results indicate that CLEC-2 facilitates motility of podocytes and retards the reconstruction of F-actin during recovery from injury. Activation of motility of podocytes may be beneficial to wound healing, but the biological significance of these phenomena needs to be clarified in in vivo studies.