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Abstract: TH-PO796

Nebulette Is a Novel Actin-Associated Protein That Stabilizes Foot Process Architecture in Kidney Podocytes

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

  • Ge, Xuhua, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Yu, Xiaoxia, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Reid, Jordan M., Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Wong, Jenny, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Bhattacharya, Smiti, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Cuttitta, Christina M., Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Campbell, Kirk N., Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Azeloglu, Evren U., Icahn School of Medicine at Mount Sinai, New York, New York, United States
Background

Nebulette (Nebl) is a mechanosensitive actin-associated protein belonging to the nebulin family that stabilizes sarcomeric structures. It has been associated with dilated cardiomyopathy, and it is thought to be cardiac-specific. Its role in kidney function is unknown. Using isobaric tagged glomarular proteomics in the puromycin-induced rat nephropathy model, we identified nebulette as the most significant glomerular protein whose expression negatively correlated with proteinuria. We hypothesized that nebulette plays a critical role in cytoskeletal stability of kidney podocytes.

Methods

We used the NephroSeq database and immunohistochemistry to check for transcript and protein expression levels of nebulette in human kidneys. Primary podocytes isolated from global nebulette knockout mice (Nebl-KO) and their wild-type littermates (WT) were used to define the role of nebulette in podocyte physiology. We used high-content microscopy, live-cell imaging, and atomic force microscopy to quantify morphological characteristics, cell motility, calcium dynamics, and cytoskeletal integrity. We evaluated nebulette's functional role in vivo using the adriamycin-induced nephropathy model in WT and Nebl-KO mice.

Results

NephroSeq database revealed that nebulette expression was enriched in healthy glomeruli and decreased significantly in DN and FSGS patients. Immunohistochemistry and immunogold electron microscopy showed localization of nebulette in human podocytes, specifically in foot processes. Morphologically, Nebl-KO podocytes showed significantly smaller spreading area, nuclear size and lower number of focal adhesions. Further, Nebl-KO podocytes exhibited significantly altered cellular motility and calcium dynamics. In vivo, there was significantly higher albuminuria and foot process effacement in Nebl-KO mice treated with adriamycin at two weeks.

Conclusion

Nebulette is a novel actin-associated protein that localizes to the foot processes of kidney podocytes, and it is associated with glomerular disease in humans. It plays a key role in podocyte physiology by stabilizing the cytoskeletal integrity and regulating focal adhesion dynamics.

Funding

  • NIDDK Support