Three-dimensional Human Kidney Tissue System for Studying Renal Diseases

描述（由申请人提供）：肾脏的主要功能是过滤肾小球中的血液，保留或消除小管中的电解质、水和其他溶质。通过疾病或毒素暴露对肾脏造成损害，挑战肾脏的功能，并最终导致肾纤维化、慢性肾脏疾病和肾衰竭。目前研究肾脏的实验室工具包括单层细胞培养和动物模型。单层细胞培养不能完全模拟人类肾脏，因为它们缺乏三维结构和组织特异性功能。动物模型不足以完全再现人类所见的损伤模式。工程化的三维（3D）培养将是体外研究肾细胞功能、功能障碍和肾毒性的一种高度相关的方法。本研究的目的是探索包含肾近端小管上皮细胞（rptec）、成纤维细胞和单核细胞的3D人肾近端小管（HRPT）组织模型，以研究组织损伤和疾病进展。3D HRPT组织模型将是一个具有预定义通道的缓慢降解的多孔丝素支架。将含有单核细胞和肾成纤维细胞的细胞外基质水凝胶注入毛细孔内。预定义的通道将支持rptec的上皮化。该模型将进行灌注培养，允许长期培养（6个月），这是对当前3D体外模型的重大改进，该模型专注于相对较短的（急性）反应。我们的假设是，3D HRPT组织模型可以作为了解肾小管功能、疾病功能障碍和毒性的工具，特别是炎症变化对损伤和修复的影响。为了解决这一假设，炎症诱导的肾纤维化和肾毒性将在工程组织中进行研究。用炎症细胞因子TNF-¿刺激组织来模拟炎症诱导的肾纤维化。肾毒素引起的小管损伤将使用环孢素、庆大霉素和顺铂进行研究。3D HRPT组织模型将允许研究急性（短期培养）和慢性（长期培养）组织反应。为了评估组织发育和损伤，我们将描述细胞活力、增殖和形态、组织结构和功能、细胞因子和细胞外基质分泌、肾损伤生物标志物、凋亡和坏死标志物。从该模型中获得的信息将为疾病病理和进展提供更好的见解。该模型还可以作为药物发现的工具，以确定治疗靶点和肾毒性。在未来，该模型可以与其他肾脏结构和炎症细胞结合，以设计更完整的肾脏，更广泛地模拟肾脏疾病。此外，该模型还可以推广到其他器官的工程中，进一步扩大其应用范围。

DESCRIPTION (provided by applicant): The kidney's primary functions are to filter blood in the glomeruli, and retain or eliminate electrolytes, water and other solutes in the tubules. Damage to the kidney, via disease or toxin exposure, challenges the function of the kidney and can eventually result in renal fibrosis, chronic kidney disease and kidney failure. Current laboratory tools to study the kidney include monolayer cell cultures and animal models. Monolayer cell cultures do not fully mimic the human kidney as they lack three-dimensional structure and tissue-specific function. Animal models are inadequate to fully reproduce the patterns of injury seen in humans. Engineered, three- dimensional (3D) culture would be a highly relevant methods of in vitro exploration of kidney cell function, dysfunction and nephrotoxicity. The goal of this research is to explore 3D human renal proximal tubule (HRPT) tissue models containing renal proximal tubule epithelial cells (RPTECs), fibroblasts and monocytes to study tissue damage and disease progression. The 3D HRPT tissue model will a slowly degrading, porous silk fibroin scaffold with predefined channels. An extracellular matrix-hydrogel containing monocytes and renal fibroblast will be infused within the pores. The predefined channels will support epithelialization by RPTECs. This model will be perfusion cultured allowing for longer term cultures (six months), a substantial improvement to current 3D in vitro models which focus on relatively short (acute) responses. Our hypothesis is that the 3D HRPT tissue models can be used as tools to understand kidney tubule function, dysfunction in disease and toxicity, specifically the effects of inflammatory changes on injury and repair. To address this hypothesis, inflammation- induced renal fibrosis and nephrotoxicity will be investigated within the engineered tissues. Inflammation- induced renal fibrosis will be modeled by stimulating tissues with the inflammatory cytokine TNF-¿. Nephrotoxin-induced tubule damage will be investigated using cyclosporine, gentamicin and cisplatin. The 3D HRPT tissue models will allow for investigation of both acute (short-term cultures) and chronic (long-term cultures) tissue responses. To assess tissue development and damage, we will characterize cell viability, proliferation and morphology, tissue structure and function, cytokine and extracellular matrix secretion, kidney injury biomarkers, and apoptosis and necrosis markers. The information garnered from this model will offer improved insight into disease pathology and progression. The model may also serve as a tool for drug discovery to identify therapeutic targets and nephrotoxicity. In the future, this model can be combined with other kidney structures and inflammatory cells to engineer a more complete kidney and more extensively model kidney diseases. Furthermore, the model can be extended to the engineering of other organs further broadening its applications.