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Increased phosphorylation of ACTN4 leads to podocyte vulnerability and proteinuric kidney disease and is stimulated by high glucose and TGF‐b

Defects in the podocyte cytoskeleton contribute to podocyte vulnerability within the glomerulus and proteinuric kidney disease. Genetic mutations in ACTN4—an essential crosslinker of the actin cytoskeleton—have been linked to proteinuric kidney disease in humans. However, how post‐translational modifications of ACTN4 affect podocyte integrity and kidney function remain unknown. We detected phosphorylation of ACTN4 at serine 159 (S159) by mass spectrometry. Our ACTN4 X‐ray crystal structure showed that phosphomimetic S159D ACTN4 changes the charge at S159 without changing the protein’s conformation. Phosphomimetic Actn4 mouse models were generated using CRISPR/Cas9 technology, and these mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. The link between ACTN4 phosphorylation at S159 and renal pathology prompted us to use targeted mass spectrometry to identify upstream signaling pathways that might increase ACTN4 phosphorylation at S159. Since high extracellular glucose and TGF‐b can both lead to kidney disease and podocyte dysfunction, we examined whether these stimuli lead to increased ACTN4 phosphorylation at S159. We found that mean S159 phosphorylation was nearly 2‐fold higher in podocytes treated with high glucose vs mannitol control (p<0.05). Similarly, mean S159 phosphorylation was nearly 2‐fold higher in the podocytes treated with TGF‐b vs in untreated podocytes (p<0.05). Moreover, we quantified the F‐actin alignment in phalloidin‐stained podocytes. WT podocytes treated with high glucose demonstrated more spatially correlated F‐actin alignment, similar to the alignment seen in phosphomimetic Actn4 podocytes but different from nonphosphorylatable Actn4 podocytes. Our findings suggest that ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation. Future studies using whole animal models and human samples should determine the extent to which the detrimental effects of high glucose on the kidney are mediated by phosphorylation of ACTN4.