Abstract: FR-PO1002

In Proximal Tubular Cells, Cyclosporine Triggers Actin Reorganization and MRTF-SRF Inhibition through Changes in Cofilin Oligomerization and Activity

Session Information

Category: Transplantation

  • 1701 Transplantation: Basic and Experimental

Authors

  • Burat, Bastien, INSERM UMR U850, Limoges University, Limoges, France
  • Marquet, Pierre, INSERM UMR U850, Limoges University, Limoges, France
  • Essig, Marie, INSERM UMR U850, Limoges University, Limoges, France
Background

Calcineurin Inhibitors, Cyclosporine A (CsA) and Tacrolimus, are the keystones of immunosuppressive regimens in solid organ transplantation. However, they induce a nephrotoxicity whose mechanisms remain widely elusive. We have previously shown that CsA affect actin organization in proximal tubular cells. Here, we explored the intracellular pathways leading to this actin reorganization and its downstream consequences.

Methods

Porcine proximal tubular LLC PK-1 cells were exposed for 24 hours to CsA (5µM), and S3R (10µM) a specific inhbibitor of cofilin phosphorylation. LLC PK-1 proteome was analyzed with iTRAQ shotgun proteomics by nano-LC-QTOF tandem mass spectrometry. Actin cytoskeleton was analyzed by TRITC-phalloidin labeling of F-actin. Cofilin oligomerization state was investigated by Western blot in non-reducing conditions after formaldehyde cross-linking Na/K-ATPase activity was quantified by colorimetric assay of inorganic phosphate. Serum response factor (SRF) activity was assessed by luciferase gene reporter assay.

Results

CsA induced a decrease in perimembranous branched F-actin meshwork with a significant decrease in Factin fluorescence positive area, (-3.3%, p < 0.0001). iTRAQ analysis showed that CsA induced a decrease in F-Actin/G-Actin ratio and a decrease in cofilin/actin ratio resulting from a global actin overexpression. Furthermore, CsA induced a 20% shift from tetramer to dimer’s forms of cofilin. These modifications of F-actin/G-actin ratio and cofilin oligomerization were associated with an inhibition of SRF activity (-56% of control activity). CsA induced a 21% inhibition of the Na/K-ATPase activity. Such inhibition has been previously demonstrated to activate cofilin. The cofilin inhibitor S3-R, which has no significant impact on F-Actin/G-Actin ratio or SRF, blocked CsA effects on actin organization and SRF activity.

Conclusion

Our results suggest that CsA deeply affects the actin cytoskeleton of proximal tubular cells through the decrease in the tetrameric, polymerizing form of cofilin. This effect favored the depolymerization activit of cofilin leading to a decrease in branched actin microfilament.This reorganization of actin cytoskeleton leads to G-Actin increase and SRF inhibition, which may trigger tubular atrophy, one of the typical lesions of CsA toxicity.