The interaction between mechanical forces and cytoskeletal impairments in podocyte mediated kidney disease

项目总结/摘要 本K 01赠款提案描述了一个为期五年的指导培训计划，旨在将冯迪博士过渡到 成为一名独立的学术研究者。冯博士获得博士学位。医学院的 在艾伦考利博士的指导下，在威斯康星州。她现在正在完成她的博士后奖学金， Martin Pollak博士的实验室，他是研究肾小球肾病遗传学的国际领导者。冯医生 她的研究重点是阐明ACTN 4突变的机制，ACTN 4是一个重要的 细胞骨架蛋白-导致一种称为局灶节段性肾小球硬化症的肾小球肾病 （FSGS）。无法更好地表征FSGS基础的足细胞功能障碍阻碍了FSGS的研究。 在广泛的免疫抑制和抗肿瘤治疗之外， 高血压治疗冯博士将她的研究重点放在突变足细胞对 它在肾小球中过滤血流时经历的机械应力。到目前为止，她已经表明， 由致病突变型ACTN 4引起的生物物理变化使足细胞变脆，表现出衰竭 的收缩力和肌动蛋白细胞骨架的破坏响应周期性拉伸。在目前的提案中， 目的1旨在进一步确定由突变型ACTN 4引起的人足细胞的受损反应，不仅是为了 拉伸，而且剪切应力。她将采用器官芯片的方法来更好地模拟这些压力 同时量化足细胞的相关生物力学和分子反应。目标2将决定 ACTN 4的翻译后磷酸化是否也损害足细胞对机械刺激的反应， 应激，使用小鼠模型和从这些小鼠分离的足细胞的生物力学研究。Aim 3计划 使用CRISPR/Cas技术生成突变的ACTN 4大鼠模型，并使用活体显微镜测量 突变体和WT肾小球内的体内机械应力。通过拟议的研究，她将学习 器官芯片方法、质谱法、超分辨率成像和活体显微术。她有 在波拉克博士的领导下组建了一个导师和顾问团队， 包括Donald Ingber博士、布鲁斯莫利托里斯博士、Hanno Steen博士、道格拉斯理查森博士以及 Roger Tung，他将提供与她的作品的翻译价值有关的建议。冯博士会花95%的时间 她在这个奖项下的时间对拟议的研究，她的培训计划包括教学课程， 在哈佛的研讨会和职业发展讲习班。该项目将使冯博士 竞争独立研究奖，为此，她计划应用她的研究结果从ACTN 4和 上述多学科的方法，以进一步研究如何在肌动蛋白为基础的细胞骨架缺陷损害 足细胞对体内经历的机械应力的反应。她的目标的实现 在哈佛的巨大资源和思想领袖的连接，坐落在独特的环境中， 一个集学术和工业于一体的城市。

Project Summary/Abstract This K01 grant proposal describes a five-year mentored training program designed to transition Dr. Di Feng to become an independent academic investigator. Dr. Feng obtained her Ph.D. at the Medical College of Wisconsin under the mentorship of Dr. Allen Cowley. She is now completing her postdoctoral fellowship in the lab of Dr. Martin Pollak, an international leader in studying the genetics of glomerular kidney disease. Dr. Feng has focused her research on elucidating the mechanism by which mutations in ACTN4 – an important cytoskeleton protein – lead to a form of glomerular kidney disease called focal segmental glomerulosclerosis (FSGS). The inability to better characterize the podocyte dysfunction that underlies FSGS has hindered the field in establishing more specific, personalized treatments beyond broad immunosuppression and anti- hypertensive therapy. Dr. Feng has focused her research on the mutant podocyte’s response to the mechanical stresses it experiences while filtering blood flow in the glomerulus. She has so far shown that the biophysical changes conferred by disease-causing mutant ACTN4 render the podocyte brittle, exhibiting failure of contractile forces and actin cytoskeleton disruption in response to periodic stretch. In the current proposal, Aim 1 seeks to further define the impaired response of human podocytes caused by mutant ACTN4, not only to stretch but also to shear stress. She will employ organ-on-a-chip methods to better simulate these stresses while quantifying the associated biomechanical and molecular responses of podocytes. Aim 2 will determine whether post-translational phosphorylation of ACTN4 also impairs the response of podocytes to mechanical stress, using mouse models and biomechanical studies of podocytes isolated from these mice. Aim 3 plans to use CRISPR/Cas technology to generate a mutant ACTN4 rat model and use intravital microscopy to measure the in vivo mechanical stresses within mutant and WT glomeruli. Through the proposed research, she will learn organ-on-a-chip methods, mass spectrometry, super-resolution imaging, and intravital microscopy. She has assembled a team of mentors and advisors under Dr. Pollak entailing leaders in these respective disciplines, including Dr. Donald Ingber, Dr. Bruce Molitoris, Dr. Hanno Steen, Dr. Douglas Richardson, as well as Dr. Roger Tung, who will provide advice related to the translational value of her work. Dr. Feng will spend 95% of her time under this award toward the proposed research, and her training plan includes didactic courses, seminars, and career development workshops at Harvard. The proposed project will make Dr. Feng competitive for independent research awards, for which she plans to apply her findings from ACTN4 and the above multidisciplinary methods to further study how defects in the actin-based cytoskeleton impair the podocyte’s response to the mechanical stresses experienced in vivo. The advancement of her goals will take place within Harvard’s vast resources and connections to thought-leaders, situated in the unique environment of Boston that integrates academics and industry.