Di Feng1,2, Mukesh Kumar3,4, Jan Muntel5, Susan B Gurley6, Gabriel Birrane7, Isaac E Stillman8,9, Lai Ding10, Minxian Wang11, Saima Ahmed3, Johannes Schlondorff8, Seth L Alper8,11, Tom Ferrante2, Susan L Marquez2, Carlos F Ng12, Richard Novak2, Donald E Ingber2,13,14,15, Hanno Steen3, Martin R Pollak1,11. 1. Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts dfeng@bidmc.harvard.edu mpollak@bidmc.harvard.edu. 2. Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts. 3. Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts. 4. F.M. Kirby Neurobiology Center, Department of Neurobiology, Boston Children's Hospital, Boston, Massachusetts. 5. Biognosys AG, Schlieren, Switzerland. 6. Division of Nephrology and Hypertension, Oregon Health & Science University, Portland, Oregon. 7. Division of Experimental Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts. 8. Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts. 9. Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts. 10. NeuroTechnology Studio, Program for Interdisciplinary Neuroscience, Brigham and Women's Hospital, Boston, Massachusetts. 11. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts. 12. Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts dfeng@bidmc.harvard.edu mpollak@bidmc.harvard.edu. 13. Vascular Biology Program, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts. 14. Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts. 15. Harvard John A. Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts.
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.
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 Actn4mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4mice 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.
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