Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis
Authors: Feng Di , Kumar Mukesh , Muntel Jan , Gurley Susan B. , Birrane Gabriel , Stillman Isaac E. , Ding Lai , Wang Minxian , Ahmed Saima , Schlondorff Johannes , Alper Seth L. , Ferrante Tom , Marquez Susan L. , Ng Carlos F. , Novak Richard , Ingber Donald E. , Steen Hanno , Pollak Martin R. 
Published: June 2020

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.

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.