PP2A as a drug target for diabetic kidney disease

PP2A作为糖尿病肾病的药物靶点

• 批准号: 10220959
• 负责人: John Cijiang He
• 金额: $ 58.95万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-21 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10220959
• 关键词: Actins; Adhesions; Affect; Affinity; Albuminuria; Attenuated; Binding; Binding Proteins; Biological Assay; Biology; Catalytic Domain; Cell Adhesion; Cells; China; Chinese Herbs; Chronic Kidney Failure; Cytoskeleton; Data; Development; Diabetes Mellitus; Diabetic Nephropathy; Diabetic mouse; Disease; Disease Progression; Docking; Drug Targeting; F-Actin; Genes; Glucose; Herbal Medicine; Human; In Vitro; Inflammation; Inflammatory; Injury; Injury to Kidney; Insulin-Dependent Diabetes Mellitus; Kidney; Kidney Diseases; Knock-out; Knockout Mice; Mass Spectrum Analysis; Mediating; Methods; Modeling; Mus; Mutation; NF-kappa B; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Paper; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Play; Protein Dephosphorylation; Protein Isoforms; Protein Subunits; Protein phosphatase; Proteins; Proteinuria; Regulation; Role; Scanning; Signaling Protein; Site; Streptozocin; Surface Plasmon Resonance; Techniques; Testing; Transgenic Organisms; Variant; Western Blotting; arctigenin; base; db/db mouse; diabetic; differential expression; drebrins; effective therapy; improved; insight; kidney cell; knock-down; migration; mouse model; mutant; novel; novel drug class; novel therapeutics; p65; phenome; podocyte; protective effect; protein activation; protein expression; protein function; protein phosphatase 2A regulatory subunit 65 kDa; scaffold; transcriptome sequencing; transcriptomics

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.

标题：PP2A 在足细胞生物学和糖尿病肾病中的作用 概括 糖尿病肾病 (DKD) 仍然是慢性肾病的主要原因，但治疗选择有限。 牛蒡甙元 (ATG) 是从传统中草药牛蒡子提取物中提取的主要成分 我们的初步研究表明，这种疗法可以重新提供保护并减少 DKD 患者的蛋白尿。 数据显示，单独使用 ATG 足以减轻小鼠的蛋白尿和足细胞损伤 1 型和 2 型糖尿病模型对糖尿病患者和对照者分离的肾小球进行转录组分析。 小鼠实验表明，ATG 治疗影响的主要途径是细胞粘附和炎症。 通过结合药物亲和力，改善培养的人足细胞的细胞粘附并抑制迁移。 响应目标稳定性 (DARTS) 技术结合质谱分析，我们鉴定了蛋白质 磷酸酶 2A (PP2A) 是培养肾细胞中最主要的 ATG 结合蛋白，这一点得到了进一步证实 此外，ATG 还增强了蛋白质印迹、计算对接和表面等离子共振测定。 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。 通过质谱法研究足细胞中的 PP2A 相互作用蛋白，确定 Drebrin-1 (DBN1) 为 F- 足细胞中 PP2A 对 DBN1 在 T335 处的去磷酸化导致细胞增加。 重要的是，小鼠足细胞特异性删除 Pp2a 会导致病情加重。 糖尿病引起的足细胞和肾小球损伤以及 ATG 介导的肾脏保护功效丧失。 此外，我们发现 PPP2R2B（PP2A 的调节亚基）在人类足细胞中独特表达 其表达在人 DKD 的肾小球中下调。 PPP2R2B 降低了 PP2A 活性，并且高葡萄糖抑制了 PPP2R2B 的表达。 Association Scan 发现了人类 PPP2R2B 基因中的几个错义变异，这些变异与 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，我们探索了 PP2A 在足细胞生物学和发病机制中发挥着关键作用 为了检验我们的假设，我们将确定 PPP2R2B 在足细胞中的调节和功能。 还研究 PPP2R2B 在 PP2A 活性、亚细胞定位和足细胞功能调节中的作用 在糖尿病条件下进行体外实验并确定是否诱导 PPP2R2B 或其患病变体表达 我们还将确定足细胞中的足细胞损伤和 DKD 进展。 通过关注 DBN1 的作用，我们将研究 PP2A 如何影响足细胞中的下游信号。 通过调节 DBN1 磷酸化来发挥足细胞功能 DBN1 的作用及其磷酸化。 还使用转基因方法对患有 DKD 的糖尿病小鼠进行了研究，这些研究将帮助我们揭示新的内容。 对足细胞生物学和 DKD 发病机制的见解，并确定 DKD 的潜在新疗法。