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

摘要 慢性肾脏疾病影响着超过2600万美国人。对于100万患有终末期肾病的患者， 和肾移植是唯一的治疗选择然而，透析是治标不治本的，肾脏捐献者供不应求。 因此，迫切需要新的治疗策略。肾的基本单位是肾单位，一个高度血管化的肾单位。 过滤和回收装置。在肾单位中，肾小球中产生的血浆滤液进入近端小管 （PT）。PT内衬立方近曲小管上皮细胞（PTECs），这是肾单位的主要吸收细胞类型 其特征在于通道和转运蛋白的极化分布，所述通道和转运蛋白从血管中回收必需的分子和离子。 血浆滤液在急性肾损伤中，PTECs非常容易受到损伤。幸存的PTEC可以修复受伤的人 肾单位，但内源性修复机制还没有得到很好的理解。这减缓了新的治疗方法的发展 加速急性和慢性肾脏疾病PT修复的策略。最近，麦克马洪实验室发现， 转录因子SOX 9在小鼠急性肾损伤后的PTECs中上调。该SOX 9 + PTEC群体 重新填充肾单位并恢复功能。然而，是否类似的机制是人类PT修复的基础， 不清楚鉴定人类PT再生机制的唯一实用方法之一是研究人类PTEC 体外培养然而，常规的培养基质是高度人工的，并且缺乏天然培养基中存在的物理线索。 影响PTEC表型和存活的PT，如流体剪切应力。因此，传统二维培养中的PTEC 失去极性和功能性。最近，已经开发了“芯片上器官”方法以将PTEC在体外暴露于 物理线索类似于那些在体内，如流体剪切应力。在这些平台内培养的PTEC形成分化的 结构，并具有改进的功能。然而，现有平台需要专用设备， 对于大多数研究小组来说，忽略了包括支持细胞群（如内皮细胞），并且没有使用 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McCain实验室在工程方面的经验， 横纹肌的“器官芯片”模型我们的关键设计参数是将流体剪切应力施加到初级人体 PTECs在蛋白质衍生的细胞外基质（ECM）水凝胶中作为小管培养， 模数在验证我们的工程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善 PTEC的活力、结构和/或功能。在目标2中，我们将对我们的工程PT诱导全局和局部损伤， 检查修复过程中整个PT中SOX 9的表达。然后我们将确定是否操纵SOX 9 活动可以增强PT修复。这个项目特别适合EBRG的资助机制，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：肾脏开发领域的知名研究者）开发一种新的工程化PT组织平台， 我们的假设驱动的研究SOX 9介导的人类PT再生机制。

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.