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：PP 2A在足细胞生物学和糖尿病肾病中的作用 总结 糖尿病肾病（DKD）仍然是慢性肾病的主要原因，治疗选择有限。 牛蒡子苷元（Arctigenin，ATG）是从中药牛蒡子提取物中提取的一种主要成分 一种已被证明具有肾保护作用并减少DKD患者蛋白尿的药物。我们的初步 数据显示单独施用ATG足以减轻小鼠的蛋白尿和足细胞损伤 1型和2型糖尿病的模型。糖尿病患者和正常人肾小球的转录组学分析 小鼠的研究表明，受ATG治疗影响的主要途径是细胞粘附和炎症。ATG 在培养的人足细胞中改善细胞粘附并抑制迁移。通过结合药物亲和性 响应目标稳定性（DARTS）技术与质谱分析，我们确定了蛋白质 磷酸酶2A（PP 2A）作为培养的肾细胞中最高的ATG结合蛋白，这一点得到了进一步证实， 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 PP 2A在培养的足细胞和糖尿病肾小球中的活性，导致p65 NF-κB的去磷酸化。 通过质谱法研究足细胞中的PP 2A相互作用蛋白，将Dreplastin-1（DBN 1）鉴定为F- 肌动蛋白相互作用蛋白在足细胞中，PP 2A使DBN 1在T335处去磷酸化，导致细胞凋亡增加。 粘附和迁移减少。重要的是，小鼠足细胞特异性Pp 2a缺失导致加重 糖尿病诱导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 此外，我们发现PP 2A的调节亚基PPP 2 R2 B在人类足细胞中独特表达， 肾小球其在人DKD肾小球中表达下调。在人类足细胞中， PPP 2 R2 B降低PP 2A活性，PPP 2 R2 B的表达被高糖抑制。全表型组 关联扫描鉴定了人类PPP 2 R2 B基因中的几种错义变体， 肾脏结局更差或更好。这些数据支持PP 2A在人类肾脏疾病中的关键作用。 基于这些观察，我们假设PP 2A在足细胞生物学和发病机制中起关键作用 的DKD。为了验证我们的假设，我们将确定PPP 2 R2 B在足细胞中的调节和功能。我们将 我们还研究了PPP 2 R2 B在PP 2A活性、亚细胞定位和足细胞功能调节中的作用 并确定PPP 2 R2 B或其患病变体表达的诱导 影响糖尿病小鼠足细胞损伤和DKD进展。我们还将确定 PP 2A在足细胞中的下游信号传导，关注DBN 1的作用。我们将研究PP 2A如何影响 足细胞通过调节DBN 1磷酸化来发挥功能。DBN 1及其磷酸化的作用将是 还使用转基因方法在患有DKD的糖尿病小鼠中进行了研究。这些研究将帮助我们揭示新的 深入了解足细胞生物学和DKD的发病机制，并确定DKD的潜在新疗法。