PP2A as a drug target for diabetic kidney disease

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Title: Role of PP2A in podocyte biology and diabetic kidney disease SUMMARY Diabetic kidney disease (DKD) remains a leading cause of chronic kidney disease with limited treatment options. Arctigenin (ATG) is a major component derived from the extracts of Fructus Arctii, a traditional Chinese herbal remedy that has shown to confer renoprotection and to reduce proteinuria in patients with DKD. Our preliminary data show that ATG administration alone is sufficient to attenuate proteinuria and podocyte injury in mouse models of type 1 and type 2 diabetes. Transcriptomic analysis of isolated glomeruli from the diabetic and control mice showed that the major pathways affected by ATG treatment were of cell adhesion and inflammation. ATG improved cell adhesion and inhibited migration in cultured human podocytes. By combining the Drug Affinity Responsive Target Stability (DARTS) technique with Mass Spectrometry analysis we identified protein phosphatase 2A (PP2A) as a top ATG-bound protein in cultured renal cells, and this was further confirmed by western blot, computational docking, and surface plasmon resonance assay. In addition, ATG enhanced the activity of PP2A in cultured podocytes and in diabetic glomeruli, resulting in dephosphorylation of p65 NF-κB. Studying the PP2A interacting proteins in podocytes by Mass Spectrometry identified Drebrin-1 (DBN1) as a F- actin interacting protein. Dephosphorylation of DBN1 at T335 by PP2A in podocytes resulted in increased cell adhesion and decreased migration. Importantly, podocyte-specific deletion of Pp2a in mice led to aggravated diabetes-induced podocyte and glomerular injury and the loss of efficacy in ATG-mediated renoprotection. In addition, we found that PPP2R2B, a regulatory subunit of PP2A, expresses uniquely in podocytes in human glomeruli. Its expression is downregulated in the glomeruli of human DKD. In human podocytes, the knockdown of PPP2R2B reduced PP2A activity and expression of PPP2R2B is suppressed by high glucose. Phenome Wide Association Scan identified several missense variants in the human PPP2R2B gene which were associated with either worse or better renal outcomes. These data support a critical role of PP2A in human kidney disease. Based on these observations, we hypothesized that PP2A plays a key role in podocyte biology and pathogenesis of DKD. To test our hypothesis, we will determine the regulation and function of PPP2R2B in podocyte. We will also study the role of PPP2R2B in the regulation of PP2A activity, subcellular localization, and podocyte function in vitro under diabetic conditions and determine whether induction of PPP2R2B or its diseased variant expression in podocytes affects podocyte injury and DKD progression in diabetic mice. We will also determine the downstream signaling of PP2A in podocytes by focusing on the role of DBN1. We will study how PP2A affects podocyte function through regulating DBN1 phosphorylation. The role of DBN1 and its phosphorylation will be also studied in diabetic mice with DKD using transgenic approach. These studies will help us to reveal new insights in the podocyte biology and the pathogenesis of DKD and identify potential new therapy for DKD.