Engineering a Kidney Organoid Model to Investigate Fibronectin-TGF-beta Signaling in Renal Fibrosis

慢性肾脏疾病(CKD)估计影响大约11%-13%的全球人口。慢性肾脏病是由肾组织的疤痕形成引起的，这个过程被称为“肾纤维化”。这一过程极大地受到由炎症细胞分泌的转化生长因子-β蛋白和纤维连接蛋白的影响，纤维连接蛋白是一种结构蛋白，由细胞组装成纤维，以建立新的组织。纤维化的过程被两个问题搞混了。首先，转化生长因子-β诱导细胞将纤维连接蛋白组装成纤维，而纤维连接蛋白纤维结合转化生长因子-β并诱导细胞对其作出反应；这种反馈使人难以区分纤维化的因果关系。其次，肾脏中多种类型的细胞可以分泌转化生长因子-β，分泌纤维连接蛋白，并将纤维连接蛋白组装成纤维，因此很难区分不同类型的细胞在纤维化进展中的作用。该项目将通过开发模仿肾组织的中空肾细胞球体来解决这一复杂性。去除/抑制特定细胞类型和/或转化生长因子-β和纤维连接蛋白信号转导元件将有助于更深入地了解这些蛋白如何促进肾纤维化。这项工作的影响将扩大，制定一个为期四个会议的研讨会，目标是来自代表不足的社区的高中生和社区大学生，重点是肾功能和细胞生物学的基础知识。慢性肾脏病是由肾脏纤维化推动的，这是一种细胞外基质过度组装的过程。免疫细胞因子转化生长因子-β和细胞外基质蛋白纤维连接蛋白在肾纤维化中的相互作用尚不完全清楚。免疫细胞会对炎症信号作出反应，释放细胞因子转化生长因子-β。转化生长因子-β促进细胞外基质蛋白纤维连接蛋白的分泌增加，并随后将纤维连接蛋白组装成纤维支架。免疫细胞来源的转化生长因子-β也促进内源性转化生长因子-β的分泌增加，从而结合到纤维连接蛋白纤维的支架上。这些因素共同推动了肾纤维化过程中肾小管形态、肾小管功能和细胞外基质重塑的改变。在这项工作中，将使用简化论者的方法来确定纤维连接蛋白纤维组装、内源性转化生长因子-β分泌和转化生长因子-β/纤维连接蛋白拴系在肾纤维化和肾损害中的作用。为了更好地了解它们各自的作用，将设计出肾小管球体，作为研究肾纤维化中转化生长因子-β/纤维连接蛋白相互作用的平台。该模拟肾组织由肾上皮细胞、成纤维细胞、周细胞以及相关的肾小管间质细胞外基质成分组成。这项工作将首先确定这些肾脏模拟物是否概括了肾小管的极性和肾小管功能，并将证明已知的肾纤维化诱因推动肾球体发生类似的变化。以下系统元素将受到干扰：i)纤维连接蛋白在细胞外基质纤维中的组装，ii)内源性转化生长因子-β的表达和分泌，以及iii)转化生长因子-β与纤维连接蛋白纤维的定位和连接，以更好地了解这些关键成分之间的相互作用。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Chronic kidney disease (CKD) is estimated to affect approximately 11-13% of the global population. CKD is driven by scarring of kidney tissue, a process known as “renal fibrosis”. This process is dramatically affected by both the protein TGF-beta, which is secreted by inflammatory cells, and the protein fibronectin, which is a structural protein assembled into fibrils by cells to build new tissue. The process of fibrosis is confounded by two issues. First, TGF-beta induces cells to assemble fibronectin into fibrils, while fibronectin fibrils bind TGF-beta and induce cells to respond to it; this feedback makes it difficult to separate cause-and-effect in fibrosis. Second, multiple cell types in the kidney can secrete TGF-beta, secrete fibronectin, and assemble fibronectin into fibrils, making it difficult to distinguish the contributions of different cell types to the progression of fibrosis. This project will address this complexity by developing hollow spheres of kidney cells that mimic kidney tissue. Removal/ inhibition of specific cell types and/or elements of TGF-beta and fibronectin signaling will allow for a deeper understanding of how these proteins contribute to renal fibrosis. The impact of this work will be expanded by developing a four-session symposium targeted to high school students and community college students from under-represented communities that focuses on the fundamentals of kidney function and cell biology.CKD is driven by renal fibrosis, which is a process of excess assembly of extracellular matrix. The interplay between the immune cytokine TGF-beta and the extracellular matrix protein fibronectin in renal fibrosis is incompletely understood. In response to inflammatory signals, the cytokine TGF-beta is released from immune cells. TGF-beta drives increased secretion of the extracellular matrix protein fibronectin and subsequent assembly of fibronectin into a scaffold of fibrils. Immune cell-derived TGF-beta also drives an increased secretion of endogenous TGF-beta, which subsequently binds to the scaffold of fibronectin fibrils. Collectively, these drive alterations in renal tubule morphology, renal tubule function, and extracellular matrix remodeling that occur during renal fibrosis. In this work, a reductionist approach will be used to identify the contribution of fibronectin fibril assembly, endogenous TGF-beta secretion, and TGF-beta/fibronectin tethering to renal fibrosis and kidney damage. To better understand their respective roles, renal tubule spheroids will be engineered that will serve as a platform for investigating TGF-beta/fibronectin interactions in renal fibrosis. This renal tissue mimetic will consist of renal epithelial cells, fibroblasts, and pericytes, as well as relevant tubulo-interstitial extracellular matrix components. The work will first determine if these renal mimetics recapitulate kidney tubule polarity and tubular function and will demonstrate that known inducers of renal fibrosis drive similar changes in the renal spheroids. The following system elements will then be perturbed: i) the assembly of fibronectin into extracellular matrix fibrils, ii) the expression and secretion of endogenous TGF-beta, and iii) the localization and tethering of TGF-beta to fibronectin fibrils to better understand the interactions between these key constituents.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.