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: 10627834
• 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/10627834
• 关键词: Actins; Adhesions; Affect; Affinity; Albuminuria; Attenuated; Binding; Binding Proteins; Biological Assay; Biology; Cell Adhesion; Cells; China; Chinese Herbs; Chronic Kidney Failure; Cytoskeleton; Data; Development; Diabetes Mellitus; Diabetic Nephropathy; Diabetic mouse; Disease; Disease Progression; Docking; Drug Combinations; 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; Persons; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Play; Protein Dephosphorylation; Protein Isoforms; Protein phosphatase; Proteins; Proteinuria; Regulation; Role; Scanning; Signaling Protein; Site; Streptozocin; Surface Plasmon Resonance; Techniques; Testing; Transgenic Organisms; Variant; Western Blotting; arctigenin; 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; type I and type II diabetes

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脏疾病中的作用 概括 糖尿病肾病（DKD）仍然是慢性肾脏疾病的主要原因，治疗方案有限。 亚氏素（ATG）是源自果汁果的提取物，这是一种传统的中草药。 已显示出对DKD患者的肾脏肾脏侵蚀并减少蛋白尿症的补救措施。我们的初步 数据表明，仅ATG给药就足以减弱小鼠的蛋白质和足细胞损伤 1型和2型糖尿病的模型。来自糖尿病和对照的分离肾小球的转录组分析 小鼠表明，受ATG治疗影响的主要途径是细胞粘附和炎症。 ATG 改善的细胞粘合剂和抑制人类足细胞中的迁移。通过结合药物亲和力 响应式靶稳定性（DARTS）技术通过质谱分析，我们确定了蛋白质 磷酸酶2a（PP2A）作为培养的肾细胞中ATG结合的顶部蛋白，这进一步证实了这一点 蛋白质印迹，计算对接和表面等离子体共振分析。另外，ATG增强了 PP2A在培养的足细胞和糖尿病肾小球中的活性，导致p65 NF-κB的磷酸化。 通过质谱法研究PP2A相互作用的蛋白质，质谱鉴定为Drebrin-1（DBN1）是F- 肌动蛋白相互作用蛋白。 PP2A在PP2A中对DBN1的去磷酸化导致细胞增加 粘附和改善的迁移。重要的是，小鼠的PP2A的足细胞特异性缺失导致聚集 糖尿病诱导的足细胞和肾小球损伤以及ATG介导的肾上腺素损伤的效率丧失。在 此外，我们发现PP2A的调节亚基PPP2R2B在人类的足细胞中独特地表达 glomerulli。它的表达在人类DKD的肾小球中被下调。在人类足细胞中，敲除 PPP2R2B的降低PP2A活性和PPP2R2B的表达被高葡萄糖抑制。现象宽 关联扫描确定了人类PPP2R2B基因中的几种错义变体 更糟或更好的肾脏结果。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，我们假设PP2A在足细胞生物学和发病机理中起关键作用 DKD。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。我们将 还研究PPP2R2B在PP2A活性，亚细胞定位和足细胞功能的调节中的作用 体外在糖尿病条件下，确定PPP2R2B的诱导还是其分离的变体表达 在足细胞中，糖尿病小鼠的足细胞损伤和DKD进展。我们还将确定 PP2A在足细胞中的下游信号传导通过重点放在DBN1的作用上。我们将研究PP2A如何影响 通过调节DBN1磷酸化的足细胞功能。 DBN1及其磷酸化的作用将是 还使用转基因方法在糖尿病小鼠中进行了研究。这些研究将帮助我们揭示新的 对足细胞生物学的见解和DKD的发病机理，并确定了DKD的潜在新疗法。

项目成果

A central role for mesangial cells in the initiation of diabetic nephropathy.

系膜细胞在糖尿病肾病的发生中起核心作用。

• DOI: 10.1016/j.kint.2023.03.033
• 发表时间: 2023
• 期刊: Kidney international
• 影响因子: 19.6
• 作者: Fang,Zhengying;Lee,Kyung;He,JohnCijiang
• 通讯作者: He,JohnCijiang