Engineering a Physiomimetic Glomerulus-on-Chip to Model Diabetic Kidney Disease

项目摘要/摘要 糖尿病肾病(DKD)影响高达40%的糖尿病患者。尽管葡萄糖的使用量在增加- 随着降压药物的使用，DKD的患病率正在上升。关节的损伤和功能障碍 肾小球中的特化上皮细胞已被证明是DKD的基本成分。 病理学。然而，DKD的治疗发现，以及肾脏疾病，都落后于其他领域 缺乏准确捕捉肾病复杂病理生理学的分析方法。芯片上的器官是 作为新的体外平台迅速崛起，用于模拟疾病和测试潜在的治疗性化合物。AS 为此目的采用芯片上的器官，需要深入的平台表征和验证 疾病模型。因此，在芯片上的肾小球中开发DKD模型将是有价值的 研究工具。在这个方案中，我们试图设计一个肾小球的仿生模型，它可以是 用来研究DKD的病理生理学。为了实现这一目标，一种彻底的工程方法将是 实施以设计和构建芯片上器官平台，以概括 肾小球滤过屏障。根据工程设计的输出，将快速成型设备 用于构建无PDMS的用于细胞培养的芯片上器官和定制的液体处理系统将被实施 重述在肾小球中发现的生理压力。一种有条件的语言的分化和培养 通过暴露于微环境应激，如细胞外，使人足细胞系永生化 矩阵刚度、化学信号和压力梯度将在平台上进行优化。这是假设的 包括这些微环境特征将增强足细胞特异性基因和 维持肾小球滤过屏障所需的蛋白质。接下来，工程化肾小球的功能- 芯片上将通过使用临床相关蛋白质的过滤试验进行评估。最后，DKD将被诱导并 以芯片上的肾小球平台为特征。疾病表型的诱导将通过以下方式完成 使细胞接触从DKD患者分离的血清。这种方法比人工的方法更有优势 损伤诱导剂，这降低了潜在的治疗性化合物对人体有益的可能性 临床背景。据推测，暴露在患者血清中将导致表型和功能两方面的结果 改变。滤过功能的改变将通过相同的滤过试验来评估，以证明正常 过滤功能。肾小球内皮细胞和足细胞之间的信号变化也将是 评估过了。最终，这项提议旨在开发一种新型的芯片上的肾小球，它将能够模拟 DKD的病理生理学。希望这个平台将有助于开发新的治疗化合物 治疗DKD。

Project Summary/Abstract Diabetic kidney disease (DKD) affects up to 40% of patients with diabetes. Despite the increasing use of glucose- and blood pressure- lowering medication, the prevalence of DKD is on the rise. Injury and dysfunction of the specialized epithelial cells in the kidney glomeruli have been shown to be a fundamental component of DKD pathology. However, therapeutic discovery for DKD, and kidney disease in general, has lagged other areas due to the lack of assays that faithfully capture the complex pathophysiology of nephropathy. Organs-on-chips are quickly emerging as novel in-vitro platforms to model diseases and test potentially therapeutic compounds. As organs-on-chips are adopted for this purpose, there is a need for in-depth platform characterization and validated disease models. Therefore, the development of a DKD model in a glomerulus-on-chip will serve as a valuable research tool. In this proposal, we seek to engineer a physiomimetic model of the glomerulus, which can be utilized to study the pathophysiology of DKD. To achieve this goal, a thorough engineering methodology will be implemented to design and build an organ-on-chip platform to recapitulate the microenvironment of the glomerular filtration barrier. Based on the output of the engineering design, rapid prototyping equipment will be used to build a PDMS-free organ-on-chip for cell culture and custom fluid handling systems will be implemented to recapitulate physiologic pressures found in the glomerulus. The differentiation and culture of a conditionally immortalized human podocyte cell line through exposure to microenvironmental stresses, such as extracellular matrix stiffness, chemical signals, and pressure gradients, will be optimized on the platform. It is hypothesized that inclusion of these microenvironmental features will enhance the expression of podocyte specific genes and proteins required for maintaining the glomerular filtration barrier. Next, functionality of the engineered glomerulus- on-chip will be assessed via a filtration assay using clinically relevant proteins. Finally, DKD will be induced and characterized on the glomerulus-on-chip platform. Induction of the disease phenotype will be accomplished by exposing cells to sera isolated from patients with DKD. This approach is more advantageous than artificial inducers of injury, which decrease the likelihood that potentially therapeutic compounds will be beneficial in a clinical context. It is hypothesized that exposure to patient sera will result in both phenotypic and functional changes. Changes in filtration function will be assessed by the same filtration assay used to demonstrate normal filtration function. Changes in signaling between glomerular endothelial cells and podocytes will also be assessed. Ultimately, this proposal aims to develop a novel glomerulus-on-chip, which will be able to model the pathophysiology of DKD. Hopefully, this platform will assist in the development of new therapeutic compounds to treat DKD.