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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的慢性肾病影响着超过 2600 万美国人，其中 100 万患有终末期肾病的患者需要透析。肾移植是唯一的治疗选择，但透析只是姑息治疗，而且肾脏捐赠者供不应求。因此，迫切需要新的治疗策略。肾脏的基本单位是肾单位，它是一个高度血管化的单位。在肾单位中，肾小球中产生的血浆滤液进入近端小管。 (PT) 内衬有立方形近端小管上皮细胞 (PTEC)，这是肾单位的主要吸收细胞类型。其特征是通道和转运蛋白的极化分布，可从细胞中回收必需的分子和离子在急性肾损伤中，PTEC 非常容易受到损伤，幸存的 PTEC 可以修复受损部位。肾单位，但内源性修复机制尚不清楚，这减缓了新疗法的开发。最近，麦克马洪实验室发现，加速急性和慢性肾脏疾病 PT 修复的策略。小鼠急性肾损伤后，PTEC 中的转录因子 SOX9 上调。PTEC 的 SOX9+ 群体。然而，人类 PT 修复是否有类似的机制存在？确定人类 PT 再生机制的唯一实用方法之一是研究人类 PTEC。然而，传统的培养基质高度人工化，缺乏天然的物理线索。影响 PTEC 表型和存活的 PT，例如流体剪切应力因此，传统二维培养中的 PTEC。最近，“芯片上的器官”方法已被开发出来，可以将 PTEC 暴露在体外。类似于体内的物理信号，例如在这些平台中培养的 PTEC 形成差异化。然而，现有平台需要无法访问的专用设备。对于大多数研究小组来说，忽略了支持细胞群（例如内皮细胞），并且尚未使用作为识别 PT 再生机制的工具，因此，在目标 1 中，我们将使用现成的设备来设计一个利用麦凯恩实验室的工程经验，用于设计和维护人类 PT 的可扩展平台我们的关键设计参数是对原始人类施加流体剪切应力。 PTEC 在弹性相对较低的蛋白质来源的细胞外基质 (ECM) 水凝胶中培养为管状在验证我们的工程 PT 概括了关键的结构和功能表型后，我们将添加支持细胞群（内皮细胞、成纤维细胞）进入 ECM 水凝胶并建立任何进一步的改进在目标 2 中，我们将诱导我们工程化的 PT 和局部损伤。在修复过程中检查整个 PT 中 SOX9 的表达，然后我们将确定是否操纵 SOX9。活动可以增强 PT 修复该项目特别适合 EBRG 融资机制，因为我们有建立了一个多学科团队（Megan McCain教授：生物医学工程初级研究员；McCain教授 Andy McMahon：肾脏发育领域的知名研究员）开发新的工程 PT 组织平台，以实现我们对 SOX9 介导的人类 PT 再生机制的假设驱动研究。