Activation of Protease-activated receptor 2 by zonulin regulates cell-cell contac

zonulin 激活蛋白酶激活受体 2 调节细胞间接触

• 批准号: 8927989
• 负责人: Karla Johanna Schramm
• 金额: $ 3.67万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8927989
• 关键词: Accounting; Actins; Adaptor Signaling Protein; Adhesions; Affect; Albumins; Albuminuria; Binding Sites; Celiac Disease; Cell Nucleus; Cell surface; Cells; Cellular biology; Chronic Kidney Failure; Clinical Research; Cytoskeleton; Diet; Dissociation; Drug Targeting; Epithelial; Epithelial Cells; Filtration; Focal Segmental Glomerulosclerosis; Foot Process; Functional disorder; G-Protein-Coupled Receptors; Gluten; Goals; Healthcare; Heme; Hemopexin; Immigration; In Vitro; Injury; Intercellular Junctions; Intestines; Kidney; Kidney Diseases; Kidney Glomerulus; Kidney Part; Knockout Mice; Lead; Left; Ligands; Link; Mediating; Molecular Genetics; Mus; Nephrotic Syndrome; Oral; PAR-2 Receptor; Patients; Peptides; Permeability; Phenotype; Phosphorylation; Population; Protein Isoforms; Proteinase-Activated Receptors; Proteins; Proteinuria; Rattus; Regulation; Renal glomerular disease; Reporting; Serine Protease; Serum; Serum Proteins; Signal Transduction; Small Intestines; Stress Fibers; Tight Junctions; Transgenic Mice; Wild Type Mouse; base; cell motility; cell type; glomerular function; in vivo; macromolecule; monolayer; mouse model; nephrin; novel; podocyte; prevent; public health relevance; receptor; research study; slit diaphragm; zonula occludens-1 protein; zonulin

DESCRIPTION (provided by applicant): Proteinuria is a cardinal sign of chronic kidney disease (CKD), which is a major health-care problem that affects millions of people worldwide. Recent advances in molecular genetics and cell biology have revealed the podocyte as the primary functional regulator of the tri-layered glomerular filter. Since podocyte foot processes (FP) and their interposed slit diaphragm (SD) form the final barrier to protein loss, podocyte injury leads to proteinuric kidney disease. The SD is a specialized dynamic cell-cell junction and contains several proteins that are also found in tight junctions (TJ) of "classic" epithelial cells Since structural features of the SD and TJ are different, the function of TJ proteins in podocytes is unclear, but most likely involves the regulation of SD function. The serum protein zonulin has been shown to induce TJ disassembly in epithelial cells of the small intestine thereby increasing paracellular flux of macromolecules across the epithelial barrier in a regulated and reversible manner. Zonulin activates protease-activated receptor (PAR) 2 and induces signaling cascades that regulate the junction protein zonula occludens (ZO)-1 and the actin cytoskeleton. Since podocytes express PAR2 and changes in ZO-1 phosphorylation and actin dynamics are established mechanisms underlying podocyte plasticity, we hypothesize that zonulin also functions as a reversible regulator of the podocyte SD and paracellular permeability in the glomerulus. Furthermore, we postulate that aberrant long-term elevation of serum zonulin leads to irreversible podocyte injury, proteinuria and eventually progressive kidney diseases. Our preliminary in vitro experiments show that zonulin treatment induces changes in the podocyte actin cytoskeleton in a PAR2-dependent manner, and also alters cell migration, ZO-1 phosphorylation and paracellular flux. Furthermore, zonulin increases albumin permeability of isolated rat glomeruli. Our in vivo experiments illustrate that mice on a high gluten diet, which increases serum zonulin levels, develop mild proteinuria. The goals for our proposed study are the following: First, we want to determine if zonulin-induced changes in the SD and paracellular permeability are PAR2-dependent. Therefore, we will replicate our in vitro studies in PAR2 knockdown podocytes, as well as in wild type podocytes in the presence of a PAR2-specific blocking peptide. Second, we will analyze if zonulin-mediated activation of PAR2 in podocytes leads to glomerular damage and albuminuria in vivo. We will analyze potential kidney phenotypes in an established transgenic mouse model with elevated serum zonulin. In parallel, we will inject a peptide mimicking zonulin's PAR2 binding site into wild type mice, and into a newly generated podocyte-specific PAR2 knockout mouse model. Since serum zonulin levels have been recently shown to be elevated in patients with CKD, and other circulating PAR2 activators like hemopexin and serine proteases have been linked to nephrotic syndrome, it is possible that PAR2 activation on podocytes is involved in a variety of glomerular diseases and that patients with nephrotic syndrome would benefit from pharmacological PAR2 inhibition.

描述（由申请人提供）：蛋白尿是慢性肾脏疾病（CKD）的主要迹象，这是一个主要的医疗保健问题，影响了全球数百万的人。分子遗传学和细胞生物学的最新进展揭示了足细胞作为三层肾小球滤波器的主要功能调节剂。由于足细胞脚步过程（FP）及其插入缝隙膜片（SD）构成了蛋白质丧失的最终障碍，因此足细胞损伤会导致蛋白尿肾脏疾病。 SD是一种专业的动态细胞 - 细胞连接，并且包含几种在“经典”上皮细胞的紧密连接（TJ）中也发现的蛋白质，因为SD和TJ的结构特征不同，Podocytes中TJ蛋白的功能尚不清楚，但很可能涉及SD功能的调节。血清蛋白齐原蛋白已被证明可以在小肠的上皮细胞中诱导TJ拆卸，从而以调节和可逆的方式增加了上皮屏障的大分子的细胞细胞通量。 Zonulin激活蛋白酶激活的受体（PAR）2，并诱导调节连接蛋白卵石闭塞（ZO）-1和肌动蛋白细胞骨架的信号传导级联反应。由于足细胞表达PAR2，并且ZO-1磷酸化和肌动蛋白动力学的变化是确定的足细胞可塑性的机制，因此我们假设扎氨蛋白也是斑点SD的可逆调节剂，并且是glomerulus中副细胞渗透性的可逆调节剂。此外，我们假设血清Zonulin的异常长期升高会导致不可逆的足细胞损伤，蛋白尿，并最终导致肾脏疾病。我们的初步体外实验表明，扎纽蛋白治疗以PAR2依赖性方式诱导足细胞肌动蛋白细胞骨架的变化，并且还改变了细胞迁移，ZO-1磷酸化和副细胞通量。此外，Zonulin增加了分离的大鼠肾小球的白蛋白渗透性。我们的体内实验表明，高麸质饮食上的小鼠会增加血清散发蛋白水平，从而产生轻度的蛋白尿。我们提出的研究的目标如下：首先，我们要确定齐索蛋白诱导的SD变化和细胞细胞渗透性是否依赖于PAR2依赖性。因此，在存在PAR2特异性阻断肽的情况下，我们将在PAR2敲低足细胞以及野生型足细胞中复制体外研究。其次，我们将分析足细胞中的Zonulin介导的PAR2激活导致肾小球损伤和体内蛋白尿。我们将在具有升高的血清齐原蛋白的已建立的转基因小鼠模型中分析潜在的肾脏表型。同时，我们将在野生型小鼠中注入一个模仿Zonulin的PAR2结合位点的肽，并将肽模仿型野生型小鼠，并将其新建的Podocyte特异性PAR2敲除小鼠模型中。 Since serum zonulin levels have been recently shown to be elevated in patients with CKD, and other circulating PAR2 activators like hemopexin and serine proteases have been linked to nephrotic syndrome, it is possible that PAR2 activation on podocytes is involved in a variety of glomerular diseases and that patients with nephrotic syndrome would benefit from pharmacological PAR2 inhibition.