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

据估计，慢性肾脏病（CKD）影响全球约11-13%的人口。CKD是由肾组织的瘢痕形成驱动的，该过程被称为“肾纤维化”。这一过程受到炎症细胞分泌的TGF-β蛋白和纤连蛋白的显著影响，纤连蛋白是一种由细胞组装成原纤维以构建新组织的结构蛋白。纤维化的过程被两个问题混淆。首先，TGF-β诱导细胞将纤连蛋白组装成原纤维，而纤连蛋白原纤维结合TGF-β并诱导细胞对其做出反应;这种反馈使得难以分离纤维化中的因果关系。其次，肾脏中的多种细胞类型可以分泌TGF-β，分泌纤连蛋白，并将纤连蛋白组装成原纤维，使得难以区分不同细胞类型对纤维化进展的贡献。该项目将通过开发模拟肾组织的肾细胞空心球体来解决这种复杂性。去除/抑制特定细胞类型和/或TGF-β和纤连蛋白信号传导的元件将允许更深入地了解这些蛋白质如何促成肾纤维化。这项工作的影响将通过开发针对高中生和社区大学生的四节专题讨论会来扩大，这些学生来自代表性不足的社区，重点关注肾功能和细胞生物学的基础知识。CKD是由肾纤维化驱动的，肾纤维化是细胞外基质过度组装的过程。免疫细胞因子TGF-β和细胞外基质蛋白纤连蛋白在肾纤维化中的相互作用还不完全清楚。作为对炎症信号的响应，细胞因子TGF-β从免疫细胞中释放。TGF-β驱动细胞外基质蛋白纤连蛋白的分泌增加，并随后将纤连蛋白组装成原纤维支架。免疫细胞衍生的TGF-β还驱动内源性TGF-β分泌增加，其随后结合到纤连蛋白原纤维的支架上。总的来说，这些驱动肾小管形态、肾小管功能和细胞外基质重塑的改变，这些改变发生在肾纤维化期间。在这项工作中，还原论的方法将被用来确定纤连蛋白原纤维组装，内源性TGF-β分泌，TGF-β/纤连蛋白拴系肾纤维化和肾损伤的贡献。为了更好地了解它们各自的作用，将设计肾小管球状体，作为研究肾纤维化中TGF-β/纤连蛋白相互作用的平台。该肾组织模拟物将由肾上皮细胞、成纤维细胞和周细胞以及相关的肾小管间质细胞外基质组分组成。这项工作将首先确定这些肾脏模拟物是否重现肾小管极性和肾小管功能，并将证明已知的肾纤维化诱导剂驱动肾球状体的类似变化。然后，以下系统元素将受到扰动：i）纤连蛋白组装成细胞外基质原纤维，ii）内源性TGF-β的表达和分泌，和iii）TGF-β 1的定位和束缚，该奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的知识分子的支持。优点和更广泛的影响审查标准。

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.