New CRISPR/Cas9 mouse model confirms association of phosphorylation of ACTN4 at serine 159 with proteinuria and focal segmental glomerulosclerosis
Authors: Feng Di , Gurley Susan , Stillman Issac , Ding Lai , Pollak Martin 
Published: June 2021

Mutations in α‐actinin‐4 (ACTN4)—an important cytoskeleton protein that binds actin—cause a form of kidney injury in humans called familial focal segmental glomerulosclerosis (FSGS). FSGS is characterized by proteinuria and eventual progression to renal failure. All of the known disease‐causing mutations result in increased ACTN4 binding affinity to actin. We previously showed that this increased actin binding was associated with biomechanical changes that left podocytes vulnerable to mechanical stress and eventual detachment. We also previously detected with mass spectrometry that ACTN4 is phosphorylated at the serine 159 site (S159) in cultured human podocytes. Moreover, phosphomimetic S159D ACTN4 protein (which mimics the effect of phosphorylation at S159) confers similar increased actin binding affinity as disease‐causing mutant ACTN4. In the current study, we hypothesized that the changes in binding affinity associated with phosphorylation of S159 may also render the podocyte vulnerable to mechanical stress and lead to eventual proteinuria and FSGS in vivo. Using CRISPR/Cas9 technology, we developed a new phosphomimetic mouse model Actn4 S160D (analogous to S159D mutation in humans). We first isolated primary podocytes from wild‐type (WT) and homozygous Actn4S160D/S160D mice. Similar to podocytes carrying disease‐causing mutant Actn4, homozygous Actn4S160D/S160D podocytes demonstrated more aligned actin filaments and increased number of focal adhesions (quantified using vinculin staining) in comparison with WT podocytes. We subjected both WT and homozygous Actn4S160D/S160D mice to subtotal nephrectomy to induce glomerular hyperfiltration and increase glomerular mechanical stress upon podocytes. We found that in comparison with WT, homozygous Actn4S160D/S/160D mice demonstrated significantly increased albuminuria at 2, 3, and 7 weeks after nephrectomy. Additionally, kidney sections were used for PAS staining and examined by a renal pathologist to assess for glomerulosclerosis. Whereas only 1% of glomeruli were found to be sclerosed in WT, 12% were sclerosed in homozygous Actn4S160D/S/160D mice (p<0.001). Our findings confirm that increased phosphorylation at S159 is pathologic, in showing that a phosphomimetic mouse model develops albuminuria and FSGS. Moreover, we show that phosphomimetic podocytes demonstrate changes in actin alignment and focal adhesion, markers of podocyte dysfunction that are shared by disease‐causing mutant ACTN4 podocytes. Altogether, these findings add to the building evidence suggesting that abnormal phosphorylation at S159 leads to changes on a protein, cellular, and whole animal level that are similar to mutant ACTN4‐mediated disease. This evidence provides new insights into the importance of a post‐translational modification affecting the podocyte cytoskeleton.Support or Funding InformationK01DK114329 and R37DK059588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.