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2600多万美国人。对于100万终末期肾病患者，透析 和肾移植是唯一的治疗选择。然而，透析是姑息的，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，它是一种高度血管化的 过滤和回收单元。在肾单位，肾小球产生的血浆滤液进入近端小管。 (PT)。甲状旁腺内衬有立方近端小管上皮细胞(PTECs)，这是肾单位的主要吸收细胞类型。 以通道和转运体的极化分布为特征的，这些通道和转运体可以从细胞中恢复必要的分子和离子 血浆滤液。在急性肾损伤中，PTECs非常容易受到损伤。存活的PTEC可以修复受伤的人 肾单位，但内源性修复机制尚不清楚。这减缓了新疗法的发展 在急慢性肾脏疾病中加速PT修复的策略。最近，麦克马洪实验室发现 转录因子SOX9在小鼠急性肾损伤后PTECs中表达上调。这群SOX9+的PTEC 重新填充肾单位并恢复功能。然而，人类PT的修复是否有类似的机制尚不清楚 不清楚。确定人PTECs再生机制的唯一实用途径之一是研究人PTECs 体外培养。然而，传统的培养基质是高度人工的，缺乏本地人存在的物理提示。 PT影响PTEC的表型和存活，如流体剪切力。因此，在传统的二维培养中，PTECs 失去极性和功能。最近，“芯片上的器官”方法已经被开发出来，使体外培养的PTECs暴露于 与体内相似的物理线索，如流体剪切力。在这些平台中培养的PTEC形成分化的 结构，并具有改进的功能。然而，现有平台需要无法访问的专用设备 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)，并未使用 作为确定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 可扩展的平台，用于设计和维护人工PT，利用麦凯恩实验室的工程经验 横纹肌的“芯片上器官”模型。我们的关键设计参数是对原始人施加流体剪应力 在弹性相对较低的蛋白质源性细胞外基质(ECM)水凝胶中培养为小管的PTECs 模数。在验证我们设计的PT概括了关键的结构和功能表型之后，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PTEC的活性、结构和/或功能中。在目标2中，我们将对我们设计的PT和 修复过程中检测SOX9在整个甲状旁腺中的表达。然后我们将确定是否操纵SOX9 活动可以增强PT修复。这个项目特别适合EBRG筹资机制，因为我们有 建立了一个多学科团队(梅根·麦凯恩教授：生物医学工程初级研究员；麦凯恩教授 Andy McMahon：肾脏开发方面的资深研究员)开发一种新的工程化PT组织平台，以使 我们对SOX9介导的人甲状旁腺再生机制的假说驱动研究。

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.