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Abstract: PO1683

C-Type Lectin-Like Receptor (CLEC) 2, the Ligand of Podoplanin, Induced Dynamic Change of F-Actin Filaments in Podocytes

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Tanaka, Keiko, Okayama Daigaku, Okayama, Okayama, Japan
  • Matsusaka, Taiji, Tokai Daigaku Igakubu Daigakuin Igaku Kenkyuka, Isehara, Kanagawa, Japan
Background

Podoplanin is intensely expressed on podocyte membrane in an evolutionally conserved manner. Podoplanin is connected to F-actins through phosphorylated (p-)ERM in podocytes. CLEC-2, the endogenous ligand of podoplanin, is highly expressed in platelets and also exists in the plasma as a soluble form. Normally, podocytes are sequestered from CLEC-2, but when the glomerular barrier is injured, podocytes can have access to CLEC-2. We studied potential actions of CLEC-2 on podocytes.

Methods

Fc-CLEC-2, a fusion of Fc and human CLEC-2 (51-229), which can bind to mouse podoplanin, was generated in HEK293 cells and purified by Protein A chromatography. The changes of podocytes were evaluated 1 hr after the treatment with Fc or Fc-CLEC-2 (10ug/mL).
StrepTagII-FLAG-CLEC-2, a fusion of double-tags and mouse CLEC-2 (51-229), was generated in HEK293 cells and purified by StrepTactin Sepharose column. Kidneys of C57BL/6 mice were perfused with 5 ug/g BW of FLAG-CLEC-2 through the aorta. Kidney samples were collected 1hr after the perfusion.

Results

Cultured podocytes treated with Fc showed an elongated morphology with numerous F-actin filaments, while podocytes with Fc-CLEC-2 showed a round shape with degradation of F-actin. Western blot revealed that pERM/ERM ratio was 0.47-fold decreased in podocytes with Fc-CLEC-2, indicating that CLEC-2 induced dephosphorylation of ERM. Immunostaining depicted abundant moesin, which we found the major ERM in cultured podocytes, around cell protrusions in podocytes with Fc, while moesin staining was reduced in podocytes with Fc-CLEC-2.
Next, we evaluated in vivo podocytes perfused with FLAG-CLEC-2. In SEM, podocytes perfused with FLAG-CLEC-2 exhibited retraction of foot processes in 18.5% of visual fields. Western blot of glomerular lysate revealed that phosphorylation of Ezrin, the major ERM in in vivo podocytes, was 0.50-fold decreased by FLAG-CLEC-2. Double immunostaining of pERM and podocalyxin confirmed dephosphorylation of ERM in podocytes.

Conclusion

These results collectively suggest that CLEC-2 induced dephosphorylation of ERM, which then caused dissociation of podoplanin from F-actin, degradation of F-actin, leading to retraction of foot processes in podocytes. Thus, leakage of CLEC-2 may exaggerate podocyte injury.

Funding

  • Government Support – Non-U.S.