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Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis

Di Feng, Mukesh Kumar, Jan Muntel, Susan B. Gurley, Gabriel Birrane, Isaac E. Stillman, Lai Ding, Minxian Wang, Saima Ahmed, Johannes Schlondorff, Seth L. Alper, Tom Ferrante, Susan L. Marquez, Carlos F. Ng, Richard Novak, Donald E. Ingber, Hanno Steen and Martin R. Pollak
JASN July 2020, 31 (7) 1479-1495; DOI: https://doi.org/10.1681/ASN.2019101032
Di Feng
1Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts
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  • ORCID record for Di Feng
Mukesh Kumar
3Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts
4F.M. Kirby Neurobiology Center, Department of Neurobiology, Boston Children’s Hospital, Boston, Massachusetts
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Jan Muntel
5Biognosys AG, Schlieren, Switzerland
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Susan B. Gurley
6Division of Nephrology and Hypertension, Oregon Health & Science University, Portland, Oregon
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Gabriel Birrane
7Division of Experimental Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
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Isaac E. Stillman
1Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
8Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
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Lai Ding
9NeuroTechnology Studio, Program for Interdisciplinary Neuroscience, Brigham and Women’s Hospital, Boston, Massachusetts
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Minxian Wang
10Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts
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Saima Ahmed
3Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts
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Johannes Schlondorff
1Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
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Seth L. Alper
1Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
10Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts
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Tom Ferrante
2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts
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Susan L. Marquez
2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts
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Carlos F. Ng
2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts
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Richard Novak
2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts
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Donald E. Ingber
2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts
11Vascular Biology Program, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts
12Department of Surgery, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts
13Harvard John A. Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts
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Hanno Steen
3Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts
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Martin R. Pollak
1Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
10Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts
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Significance Statement

Although genetic mutations in α-actinin-4 (ACTN4) are linked with proteinuric glomerulosclerosis in humans, the effect of post-translational modifications is unknown. The authors show that ACTN4—an actin crosslinking cytoskeletal protein—is phosphorylated at serine 159 (S159) in podocytes. Compared with wild-type ACTN4, phosphomimetic ACTN4 protein demonstrated increased binding affinity to F-actin, and phosphomimetic mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress compared with controls. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. These biochemical, cellular, and renal effects are similar to those seen in mutant ACTN4-mediated proteinuric glomerulosclerosis. High extracellular glucose and TGF-β levels stimulate ACTN4 phosphorylation. These findings suggest that, in addition to genetic mutations, increased phosphorylation of ACTN4 may mediate podocyte injury and kidney disease.

Abstract

Background Genetic mutations in α-actinin-4 (ACTN4)—an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes—have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known.

Methods Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF-β levels increase phosphorylation of ACTN4.

Results Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF-β stimulates phosphorylation of ACTN4 at S159 in podocytes.

Conclusions These findings suggest that 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. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.

  • chronic kidney disease
  • focal segmental glomerulosclerosis
  • glomerular disease
  • cytoskeleton
  • podocyte
  • proteinuria
  • Copyright © 2020 by the American Society of Nephrology
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Journal of the American Society of Nephrology: 31 (7)
Journal of the American Society of Nephrology
Vol. 31, Issue 7
July 2020
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Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis
Di Feng, Mukesh Kumar, Jan Muntel, Susan B. Gurley, Gabriel Birrane, Isaac E. Stillman, Lai Ding, Minxian Wang, Saima Ahmed, Johannes Schlondorff, Seth L. Alper, Tom Ferrante, Susan L. Marquez, Carlos F. Ng, Richard Novak, Donald E. Ingber, Hanno Steen, Martin R. Pollak
JASN Jul 2020, 31 (7) 1479-1495; DOI: 10.1681/ASN.2019101032

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Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis
Di Feng, Mukesh Kumar, Jan Muntel, Susan B. Gurley, Gabriel Birrane, Isaac E. Stillman, Lai Ding, Minxian Wang, Saima Ahmed, Johannes Schlondorff, Seth L. Alper, Tom Ferrante, Susan L. Marquez, Carlos F. Ng, Richard Novak, Donald E. Ingber, Hanno Steen, Martin R. Pollak
JASN Jul 2020, 31 (7) 1479-1495; DOI: 10.1681/ASN.2019101032
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Keywords

  • chronic kidney disease
  • focal segmental glomerulosclerosis
  • glomerular disease
  • cytoskeleton
  • podocyte
  • proteinuria

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