Establishing Mechanisms of Human Proximal Tubule Regeneration in an Engineered Organ on Chip Platform

在芯片平台上的工程器官中建立人类近端小管再生机制

• 批准号: 9437497
• 负责人: ANDREW P. MCMAHON
• 金额: $ 22.84万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-23 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9437497
• 关键词: 3D Print; Ablation; Acute; Acute Renal Failure with Renal Papillary Necrosis; Adopted; Affect; Albumins; American; Biocompatible Materials; Biomedical Engineering; Biomimetics; Blood Vessels; Blood capillaries; Cell Culture Techniques; Cell Survival; Cell physiology; Cellular Structures; Chronic Kidney Failure; Cilia; Collaborations; Cues; Cultured Cells; D Cells; Data; Development; Dialysis procedure; Doxycycline; End stage renal failure; Endothelial Cells; Engineering; Epithelial Cells; Epithelium; Equipment; Extracellular Matrix; Faculty; Fibroblasts; Filtration; Foundations; Funding Mechanisms; Funding Opportunities; Future; Gelatin; Goals; Human; Human Engineering; Hydrogels; In Vitro; Injectable; Injury; Ions; Kidney; Kidney Diseases; Kidney Transplantation; Lasers; Liquid substance; Mediating; Microfabrication; Microfluidics; Modeling; Modulus; Mus; Natural regeneration; Nephrons; Nephrotoxic; Organ; Outcomes Research; Patients; Perfusion; Pharmaceutical Preparations; Phenotype; Physiological; Plasma; Plasticizers; Population; Proteins; Recovery; Regenerative Medicine; Research; Research Personnel; Research Project Grants; Role; Site; Striated Muscles; Stromal Cells; Structure; Supporting Cell; System; Testing; Therapeutic; Tissue Engineering; Tissues; Tubular formation; Work; absorption; base; capillary; cell type; design; experimental study; functional restoration; improved functioning; in vivo; injured; insight; laboratory experience; member; multidisciplinary; neglect; nephrogenesis; nephrotoxicity; novel; novel therapeutic intervention; novel therapeutics; palliative; pressure; prevent; repaired; shear stress; stem cell biology; success; therapeutic development; tool; transcription factor

Abstract Chronic kidney disease affects over 26 million Americans. For the one million patients with end stage renal disease, dialysis and kidney transplant are the only therapeutic options. However, dialysis is palliative and kidney donors are in short supply. Thus, there is a critical need for new therapeutic strategies. The basic unit of the kidney is the nephron, a highly vascularized filtration and recovery unit. In the nephron, the plasma filtrate generated in the glomerulus passes into the proximal tubule (PT). The PT is lined by cuboidal proximal tubule epithelial cells (PTECs), the major resorptive cell type of the nephron characterized by the polarized distribution of channels and transporters that recover essential molecules and ions from the plasma filtrate. In acute kidney injury, PTECs are highly susceptible to damage. Surviving PTECs can repair the injured nephron, but endogenous repair mechanisms are not well-understood. This slows the development of new therapeutic strategies to accelerate PT repair in both acute and chronic kidney disease. Recently, the McMahon lab identified that the transcription factor SOX9 is up-regulated in PTECs after acute kidney injury in mice. This SOX9+ population of PTECs repopulates the nephron and restores function. However, whether a similar mechanism underlies repair of the human PT is unclear. One of the only practical approaches to identify mechanisms of human PT regeneration is to study human PTECs cultured in vitro. However, conventional culture substrates are highly artificial and lack physical cues present in the native PT that impact PTEC phenotype and survival, such as fluid shear stress. As a result, PTECs in conventional 2-D culture lose polarity and functionality. Recently, “Organ on Chip” approaches have been developed to expose PTECs in vitro to physical cues similar to those in vivo, such as fluid shear stress. PTECs cultured within these platforms form differentiated structures and have improved functionality. However, existing platforms require specialized equipment that is not accessible to most research groups, neglect to include supporting cell populations (such as endothelial cells), and have not been used as tools for identifying mechanisms of PT regeneration. Thus, in Aim 1, we will use off-the-shelf equipment to engineer a scalable platform for engineering and maintaining a human PT, leveraging the McCain lab’s experience in engineering “Organ on Chip” models of striated muscle. Our key design parameters are to apply fluid shear stress to primary human PTECs cultured as a tubule within a protein-derived extracellular matrix (ECM) hydrogel with relatively low elastic modulus. After validating that our engineered PT recapitulates key structural and functional phenotypes, we will add supporting cell populations (endothelial cells, fibroblasts) into the ECM hydrogel and establish any further improvements in PTEC viability, structure, and/or function. In Aim 2, we will induce global and local injury to our engineered PT and examine the expression of SOX9 throughout the PT during repair. We will then determine whether manipulating SOX9 activity can augment PT repair. This project is especially well-suited for the EBRG funding mechanism because we have established a multidisciplinary team (Prof. Megan McCain: junior investigator in biomedical engineering; Prof. McCain Andy McMahon: established investigator in kidney development) to develop a new engineered PT tissue platform to enable our hypothesis-driven research into SOX9-mediated mechanisms of human PT regeneration.

抽象的 慢性肾脏疾病影响超过2600万美国人。对于一百万患有末期肾脏疾病的患者，透析 肾脏移植是唯一的治疗选择。但是，透析是姑息性的，肾脏供体供应不足。 这是对新的治疗策略的迫切需求。肾脏的基本单位是肾单位，一种高度血管化 过滤和恢复单元。在肾单位中，肾小球中产生的血浆滤波器进入代理管 （pt）。 PT由Cuboidal Tubele上皮细胞（PTEC）衬里，这是肾单位的主要吸收细胞类型 以通道和转运蛋白的极化分布为特征，从 血浆过滤器。在急性肾脏损伤中，PTEC非常容易受到损害。幸存的PTEC可以修复受伤的 肾单位，但内源性修复机制并不理解。这减慢了新疗法的发展 急性和慢性肾脏疾病中PT修复的策略。最近，麦克马洪实验室确定 小鼠急性肾损伤后PTEC中的转录因子SOX9在PTEC中上调。 Sox9+ PTEC人群 重新启动肾单位并恢复功能。但是，类似的机制是否是人类PT修复的基础 不清楚。识别人类PT再生机制的唯一实际方法之一是研究人类PTEC 体外培养。但是，传统的培养基材是高度人为的，并且缺乏本地的物理线索 影响PTEC表型和存活的PT，例如流体剪应力。结果，常规2-D培养中的PTEC 失去极性和功能。最近，已经开发了“芯片上的器官”方法，以使PTEC在体外暴露于 与体内类似的物理线索，例如流体剪切应力。在这些平台中培养的PTEC形成差异化 结构并具有改善的功能。但是，现有平台需要无法访问的专用设备 对于大多数研究小组，忽略包括支持细胞群体（例如内皮细胞），尚未使用 作为识别PT再生机制的工具。在AIM 1中，我们将使用现成的设备来设计 可扩展的平台用于工程和维护人类PT，利用麦凯恩实验室的工程经验 条纹肌肉的“芯片上的器官”模型。我们的关键设计参数是将流体剪应力施加到原发性人 PTEC在蛋白质衍生的细胞外基质（ECM）水凝胶中培养为小管，具有相对较低的弹性 模量。在验证我们的工程PT概述了关键的结构和功能表型之后，我们将补充 支持细胞群（内皮细胞，成纤维细胞）进入ECM水凝胶，并建立进一步的改进 在PTEC生存力，结构和/或功能中。在AIM 2中，我们将对我们的工程PT造成全球和地方伤害 在修复过程中检查整个PT的Sox9的表达。然后，我们将确定是否操纵Sox9 活动可以增加PT修复。该项目特别适合EBRG资金机制，因为我们有 建立了一个多学科团队（梅根·麦凯恩教授：生物医学工程的初级研究员；麦凯恩教授 安迪·麦克马洪（Andy McMahon）：肾脏发展领域的研究员）开发一个新的工程PT纸巾平台以启用 我们对人类PT再生的Sox9介导的机制的假设驱动的研究。