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Kidney Week

Abstract: PO1587

Increased Phosphorylation of ACTN4 Leads to Podocyte Dysfunction and Focal Segmental Glomerulosclerosis Mimicking Disease-Causing Mutations

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Feng, Di, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts, United States
  • Kumar, Mukesh, Boston Children's Hospital, Boston, Massachusetts, United States
  • Muntel, Jan, Biognosys AG, Schlieren, Zurich, Switzerland
  • Gurley, Susan B., Oregon Health & Science University, Portland, Oregon, United States
  • Birrane, Gabriel, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts, United States
  • Stillman, Isaac Ely, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts, United States
  • Ding, Lai, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Wang, Minxian, Broad Institute of Harvard and MIT, Boston, Massachusetts, United States
  • Ahmed, Saima, Boston Children's Hospital, Boston, Massachusetts, United States
  • Schlondorff, Johannes S., Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts, United States
  • Alper, Seth L., Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts, United States
  • Ferrante, Thomas, Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, United States
  • Marquez, Susan, Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, United States
  • Ng, Carlos F., Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, United States
  • Novak, Richard, Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, United States
  • Ingber, Donald E., Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, United States
  • Steen, Hanno, Boston Children's Hospital, Boston, Massachusetts, United States
  • Pollak, Martin R., Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts, United States
Background

Genetic mutations in ACTN4 have been linked to focal segmental glomerulosclerosis (FSGS) in humans through cytoskeletal disruption and impairment in podocyte response to mechanical stress. ACTN4 is phosphorylated at S159 in podocytes, but the effect of this post-translational modification on podocyte and kidney function is not known.

Methods

We used phosphomimetic ACTN4 to investigate the effects of this phosphorylation in vitro and in vivo. The effect of phosphorylation on the interaction between ACTN4 and F-actin was assessed through F-actin bundling assays, and the effect on F-actin alignment was assessed by immunofluorescence staining and quantified using autocorrelation analysis. Microfluidic organ-on-a-chip technology was used to measure the rate of podocyte detachment when simultaneously exposed to fluid flow and cyclic strain. A phosphomimetic mouse model was generated, subjected to subtotal nephrectomy (to simulate glomerular hyperfiltration), and assessed for renal injury. Targeted mass spectrometry was used to determine whether injurious stimuli to podocytes increased ACTN4 phosphorylation.

Results

Compared to wild type (WT) ACTN4, phosphomimetic ACTN4 led to increased F-actin bundling activity and higher spatial correlation of F-actin alignment in podocytes. When subjected to mechanical stress in organ-on-a-chip culture devices, phosphomimetic podocytes demonstrated nearly a 3-fold higher rate of detachment (28/154 podocytes, 18.2%) in comparison with WT (12/170 podocytes, 7.1%, p<0.05). Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Finally, phosphorylation of ACTN4 at S159 in podocytes was stimulated by high extracellular glucose and TGF-b.

Conclusion

Increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. Stimulation of this phosphorylation by glucose and TGF-B suggests potential mechanisms of ACTN4-mediated kidney disease that extend beyond its original genetic etiology.

Funding

  • NIDDK Support