PP2A as a drug target for diabetic kidney disease

PP2A在足细胞生物学和糖尿病肾病中的作用 摘要 糖尿病肾病(DKD)仍然是慢性肾脏疾病的主要原因，治疗选择有限。 牛至皂苷元(Arctigenin，ATG)是从传统中草药牛至的提取物中提取的主要成分。 对DKD患者给予肾脏保护和减少蛋白尿的药物。我们的预赛 数据显示，单独给予ATG足以减轻小鼠的蛋白尿和足细胞损伤 1型和2型糖尿病模型。糖尿病患者和对照组分离肾小球的转录转录分析 小鼠实验表明，ATG作用的主要途径是细胞黏附和炎症。ATG 改善培养的人足细胞的细胞黏附和抑制迁移。通过结合药物亲和力 反应靶标稳定性(DARTS)技术与质谱学分析我们鉴定了蛋白质 在培养的肾细胞中，磷酸酶2A(PP2A)是一种顶级的ATG结合蛋白，这一点被进一步证实 蛋白质印迹、计算对接和表面等离子体共振分析。此外，ATG还增强了 在培养的足细胞和糖尿病肾小球中，PP2A的活性导致p65 NF-κB去磷酸化。 用质谱法研究足细胞中的PP2A相互作用蛋白，确定Drebrin-1(DBN1)是一种F- 肌动蛋白相互作用蛋白。足细胞中PP2A对T335位DBN1的去磷酸化导致细胞数量增加 粘附性和迁移性减少。重要的是，小鼠足细胞特异性PP2A的缺失导致病情加重 糖尿病引起的足细胞和肾小球损伤以及ATG介导的肾保护作用的丧失。在……里面 此外，我们还发现PPP2R2B是PP2A的一个调节亚基，在人类足细胞中有独特的表达 肾小球。它在人DKD的肾小球中表达下调。在人类足细胞中，击倒 PPP2R2B降低PP2A活性，高糖抑制PPP2R2B表达。Phenome Wide 关联扫描发现人类PPP2R2B基因中的几个错义变体与 肾脏结果要么更差，要么更好。这些数据支持PP2A在人类肾脏疾病中的关键作用。 基于这些观察，我们推测PP2A在足细胞生物学和发病机制中起关键作用 DKD的一部分。为了验证我们的假设，我们将确定PPP2R2B在足细胞中的调节和功能。我们会 同时研究PPP2R2B在调节PP2A活性、亚细胞定位和足细胞功能中的作用 在体外糖尿病条件下并确定是否诱导PPP2R2B或其病变变体的表达 对糖尿病小鼠的足细胞损伤和DKD进展有影响。我们还将确定 通过关注DBN1的作用在足细胞中PP2A的下游信号转导。我们将研究PP2A如何影响 足细胞通过调节DBN1的磷酸化而发挥作用。DBN1及其磷酸化的作用将是 也用转基因方法研究了糖尿病小鼠的DKD。这些研究将帮助我们揭示新的 洞察足细胞生物学和DKD的发病机制，并确定潜在的DKD新疗法。

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.