Role of Par1 Polarity Proteins in Podocyte Development and Glomerular Disease

描述(由申请者提供)：本建议书描述了一个五年计划，旨在发展足细胞生物学和肾小球疾病的学术生涯。PI已经完成了由NIH支持的儿科和发育肾脏病的结构化奖学金培训计划，现在将通过独特的跨部门资源整合来扩展她的科学技能。这项计划将促进利用分子遗传学和细胞生物学的专业知识来研究在肾小球疾病背景下维持足细胞极性的机制。Katalin Susztak博士和Anne Muesch博士将指导PI的科学发展。她的主要导师Susztak博士是内科/肾脏病副教授，在确定肾小球疾病的新机制方面是该领域的领导者。内部联合导师Muesch博士是发育和分子生物学副教授，是建立细胞极性的机制方面的专家，这将在肾小球疾病的背景下进行研究。此外，为了扩大PI的训练范围并促进她对足细胞极性发展的研究，PI正在接受来自外部顾问Jonathan Barasch博士的胚胎肾脏培养实践培训，他位于附近的哥伦比亚大学。Susztak博士、Muesch博士、Barasch博士和其他几位备受尊敬的发育肾病专家将组成一个咨询委员会，提供科学和职业建议。研究重点将集中在顶端-基底端极性蛋白Par1a/b在肾脏发育过程中建立足细胞极性以及在肾小球疾病背景下维持足细胞极性方面的作用。最近在她的导师的指导下进行的研究表明，Par1a/b在发育中的肾单位以及啮齿动物和人类的足细胞中都有表达。在培养的足细胞中，对Par1a/b功能的显性负抑制可引起细胞形态的改变和裂隙隔蛋白的表达改变，这些都是支持肾小球滤器结构和功能的关键足细胞成分。在糖尿病肾病、肾病综合征和肾小球硬化的啮齿动物模型中，肾小球Par1a/b的表达发生改变。拟议的实验将使用活体小鼠模型对多西环素诱导的足细胞特异性抑制Par1a/b功能进行研究，这将使研究Par1a/b在肾单位发育(胚胎发生期间的诱导)和成年小鼠中的功能。利用腺病毒感染抑制胚胎肾培养中Par1a/b功能的载体，可在体外进一步研究Par1a/b的功能。此外，Par1a/b在肾病综合征和肾小球硬化的实验模型中的表达，使用大鼠的嘌呤霉素氨基核苷肾病(PAN)，以及在人类肾病综合征中，使用收集的对照和患病的人肾脏标本。具体目标包括：检测Par1a/b在肾单位发育过程中对足细胞分化的作用，2)检测Par1a/b在维持极化足细胞结构和肾小球滤过功能中的作用，以及3)检测Par1a/b在人和肾病综合征及肾小球硬化实验模型中的表达。 公共卫生相关性：慢性和终末期肾病(CKD和ESKD)导致显著的发病率和死亡率。我们建议研究新的途径，以深入了解蛋白尿肾病和局灶性肾小球硬化(FSGS)的机制，FSGS是儿童CKD和ESKD的主要原因。我们将研究Par1a/b在建立和维持足细胞结构中的作用，并确定极性蛋白在人类肾病综合征和肾小球硬化中的表达，为进行性肾脏疾病的治疗干预或生物标志物寻找新的靶点。好了！

DESCRIPTION (provided by applicant): This proposal describes a five year program for development of an academic career in podocyte biology and glomerular disease. The PI has completed an NIH-supported, structured fellowship training program in Pediatric and Developmental Nephrology and will now expand upon her scientific skills through a unique integration of interdepartmental resources. This program will promote expertise in use of molecular genetics and cell biology to investigate mechanisms that maintain podocyte polarity in the setting of glomerular disease. Drs. Katalin Susztak and Anne Muesch will mentor the PI's scientific development. Her primary mentor, Dr. Susztak, is an Associate Professor of Internal Medicine/ Nephrology and a leader in her field in identifying novel mechanisms of glomerular disease. The internal co-mentor, Dr. Muesch, an Associate Professor in Development and Molecular Biology, is an expert in mechanisms that establish cell polarity, which will be examined in the context of glomerular disease. In addition, to expand the PI's repertoire and facilitate her study of the development of podocyte polarity, the PI is receiving hands-on training in embryonic kidney culture from an external advisor, Dr. Jonathan Barasch, who is located nearby at Columbia University. Drs. Susztak, Muesch, Barasch and several other highly regarded developmental nephrologists will form an advisory committee to provide scientific and career advice. Research will focus on the role of apical-basal polarity proteins Par1a/b in establishing podocyte polarity during kidney development and in maintaining podocyte polarity in the setting of glomerular disease. Recent work performed under the guidance of her mentors demonstrated that Par1a/b is expressed in developing nephrons and in rodent and human podocytes. Dominant negative suppression of Par1a/b function in cultured podocytes induced changes of cell shape and altered expression of slit diaphragm proteins, which are key podocyte components that support glomerular filter structure and function. Glomerular Par1a/b expression was altered in rodent models of diabetic nephropathy, nephrotic syndrome and glomerulosclerosis. The proposed experiments will entail doxycycline-inducible, podocyte-specific suppression of Par1a/b function using an in vivo mouse model, which will allow study of Par1a/b function during nephron development (induction during embyrogenesis) and in adult mice. Adenoviral infection of constructs suppressing Par1a/b function in embryonic kidney culture will be used to further study the function of Par1a/b in vitro. In addition, Par1a/b expression in an experimental model of nephrotic syndrome and glomerulosclerosis, using puromycin aminonucleoside nephrosis (PAN) induced in rats, and in human nephrotic syndrome, using a collection of control and diseased human kidney specimens. Specific aims include: Examine Par1a/b function during nephron development on podocyte differentiation, 2) Examine Par1a/b function in maintaining a polarized podocyte structure and glomerular filter function, and 3) Examine expression of Par1a/b in human and experimental models of nephrotic syndrome and glomerulosclerosis. PUBLIC HEALTH RELEVANCE: Chronic and end-stage kidney disease (CKD and ESKD) results in significant morbidity and mortality. We propose to examine novel pathways that may provide insight into mechanisms of proteinuric kidney disease and focal glomerulosclerosis (FSGS), a leading cause of childhood CKD and ESKD. We will examine the role of Par1a/b in establishing and maintaining podocyte structure and define polarity protein expression in human nephrotic syndrome and glomerulosclerosis, potentialy identifying new targets for therapeutic intervention or biomarkers for progressive kidney disease. !