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Abstract: TH-PO737

14-3-3 Proteins Stabilize Vimentin and Actin Filaments to Maintain Primary and Foot Processes in Podocyte

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Yasuda, Hidenori, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Fukusumi, Yoshiyasu, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Zhang, Ying, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
  • Kawachi, Hiroshi, Department of Cell Biology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Background

14-3-3 proteins are a ubiquitously expressed family of adaptor proteins. Despite exhibiting high sequence homology, several 14-3-3 isoforms have isoform-specific binding partners and roles. We reported that 14-3-3β interacts with FKBP12, a binding protein of tacrolimus, and synaptopodin (Syp) to maintain the structure of actin fibers in podocytes (Yasuda et al., FASEB J, 2021). However, the functions of 14-3-3 proteins in podocyte are not fully elucidated. The precise localization and differential role of 14-3-3 isoforms in glomeruli are also unclear.

Methods

mRNA expression of 14-3-3s in kidneys was analyzed with RT-PCR, and their localization was investigated with immunohistochemical analyses. The interaction of 14-3-3s was analyzed by immunoprecipitation assay with the lysate of cultured podocytes and HEK-transfected cells. The effect of 14-3-3 isoform-specific siRNAs was analyzed in cultured podocytes. The expression of 14-3-3s in podocyte injury models was analyzed.

Results

14-3-3β and 14-3-3σ were abundantly expressed in glomeruli. 14-3-3β in glomeruli was restricted in podocytes. 14-3-3σ in glomeruli was expressed in podocytes and mesangial cells. 14-3-3β was dominantly co-localized with FKBP12 in the foot processes, and a part of 14-3-3β was co-localized with Par3 at the slit diaphragm. 14-3-3β interacted with Par3, and the interaction was decreased by the transfection of FKBP12. The interaction of Par3-Par6 was enhanced by the treatment of 14-3-3β siRNA (3 times to normal, p<0.05). The structure of F-actin was deranged (score 1.8 vs. 3.1 of normal, p<0.05), and process formation was impaired (46.5% to normal, p<0.005) in the podocytes treated with 14-3-3β siRNA. 14-3-3β and Syp expression was decreased in podocyte injury models. 14-3-3σ in podocytes was expressed in the primary processes. 14-3-3σ interacted with vimentin but not with the actin-associated proteins FKBP12 and Syp. The structure of vimentin fibers was deranged (65.2% to normal, p<0.05), and process formation was impaired (40.1% to normal, p<0.005) in the podocytes with 14-3-3σ siRNA. 14-3-3σ and vimentin expression was increased in the early phase of podocyte injury but was decreased in the late stage.

Conclusion

14-3-3 proteins play the roles in maintaining the primary and foot processes by stabilizing vimentin and actin filaments in podocyte.

Funding

  • Government Support – Non-U.S.