Synthetic approaches to study cell polarity related kidney defects

研究细胞极性相关肾脏缺陷的综合方法

基本信息

批准号：
10272419
负责人：
Louis Skjei Prahl
金额：
$ 6.98万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10272419
关键词：
3-Dimensional Address Adult Affect Automobile Driving Behavior Biochemical Blood Circulation Caliber Cell Polarity Cells Child Childhood Chronic Kidney Failure Complex Congenital Abnormality Cues DNA Defect Development Developmental Process Dimensions Disease Duct (organ) structure Dysplasia Embryo End stage renal failure Engineering Epithelial Epithelial Cells Failure Fatty acid glycerol esters Fluorescence Future Genes Genetic Goals Growth Health Human Kidney Kidney Diseases Knock-out Lead Life Ligands Logic Loose connective tissue Lung Measures Mesenchymal Mesenchyme Metanephric Diverticulum Modeling Molecular Genetics Morphology Movement Mus Mutation Neuroglia Organoids Outcome Pattern Phosphotransferases Physiological Population Positioning Attribute Process Production Proteins Protocols documentation Renal tubule structure Reporter Role Shapes Signal Pathway Signal Transduction Standardization Structure Syndrome System Technology Testing Time Tissue Engineering Tissues Trees Tube Ureter Urinary tract Urine Waste Products base cell type collecting tubule structure congenital anomalies of the kidney genetic approach high-throughput drug screening human disease human model improved in vivo induced pluripotent stem cell kidney cell mathematical model nephrogenesis neurotrophic factor novel therapeutics planar cell polarity prevent response tissue support frame tool

项目摘要

ABSTRACT Congenital abnormalities of the kidney and urinary tract (CAKUT) account for ~50% of childhood chronic kidney disease cases. Many CAKUT defects involve imprecise sizing or spacing of kidney collecting duct structures, such as duplicated ureters, cystic collecting ducts, and renal hypoplasia, which can lead to severe health problems including end-stage kidney disease. As such, there is a critical need to understand how developmental processes promote proper sizing, spacing, and positioning of kidney structures, so that CAKUT defects can be corrected. Kidney development begins through tree-like outgrowth of the ureteric bud epithelium (the future collecting duct network) into a loose connective tissue or mesenchyme. Precisely positioning ureteric bud tubules within this network requires tight control of Ret kinase signaling. Ret responds to secreted glial cell-derived neurotrophic factor (GDNF) from surrounding mesenchymal cells and mutations that affect Ret-GDNF signaling cause CAKUT defects. While Ret-GDNF signaling drives the proliferative expansion of ureteric bud epithelial cells, emerging evidence suggests that “planar cell polarity” (PCP) – a mechanism by which cells sense their planar positions within sheets and tubes – controls the shape of the collecting duct network. Significantly, recent findings indicate that mesenchymal cells expressing the PCP genes Fat4 and Dchs1 prevent improper ureteric bud branching and mutations in these genes cause CAKUT defects in mice and humans. However, it is unclear how interfaces between PCP-expressing mesenchymal cells and Ret-expressing epithelial cells enforce the precise sizing and spacing of collecting duct tubules and avoid defects. The objective of this proposal is to create controlled spatial interfaces between PCP-expressing cells and Ret-expressing cells and study their impact on Ret-GDNF signaling levels and the resultant effect on the size and shape of epithelial structures. Aim 1 of this proposal establishes a DNA-based cell patterning technology that enables the production of cell interfaces in engineered tissues. This cell patterning technology will be used to create interfaces between PCP-expressing cells and Ret-expressing cells. Cells expressing fluorescence-based kinase activity reporters and mathematical modeling will be used to study how these spatial interfaces influence Ret-GDNF signaling. Aim 2 of this proposal will pattern the two cell types in 3D tissue scaffolds and study the effects of interfaces on the size and shape of resulting epithelial structures. The central hypothesis of this proposal is that PCP-expressing cells locally restrict Ret-GDNF-driven epithelial tissue growth at interfaces, thereby producing structures of defined size and shape. Together, the approaches developed in this proposal will improve our understanding of the cellular mechanisms that cause CAKUT defects and create new tools to build defined tissue structures in organoid models of human disease.

抽象的先天性肾脏和泌尿道异常 (CAKUT) 约占儿童慢性病的 50% 许多 CAKUT 缺陷涉及肾集合管的尺寸或间距不精确。结构，如重复的输尿管、囊性集合管和肾发育不全，这可能导致严重的因此，迫切需要了解如何解决包括终末期肾病在内的健康问题。发育过程促进肾脏结构的适当大小、间距和定位，以便 CAKUT 肾脏发育的缺陷可以通过输尿管芽的树状生长开始。上皮（未来的集合管网络）变成疏松的结缔组织或间质。将输尿管芽小管定位在该网络内需要严格控制 Ret 激酶 Ret 反应。从周围的间充质细胞分泌胶质细胞源性神经营养因子（GDNF）和突变 Ret-GDNF 信号传导导致 CAKUT 缺陷，而 Ret-GDNF 信号传导则驱动增殖。随着输尿管芽上皮细胞的扩张，新出现的证据表明“平面细胞极性”（PCP）—— 细胞感知其在片材和管内的平面位置的机制——控制形状值得注意的是，最近的研究表明间充质细胞表达 PCP。 Fat4 和 Dchs1 基因可防止输尿管芽分支不当，这些基因的突变会导致 CAKUT 然而，尚不清楚表达 PCP 的间充质细胞之间如何相互作用。细胞和表达 Ret 的上皮细胞强制集合管小管的精确尺寸和间距，该提案的目的是在 PCP 表达之间创建受控的空间界面。细胞和 Ret 表达细胞，并研究它们对 Ret-GDNF 信号水平的影响以及由此产生的影响该提案的目标 1 建立了基于 DNA 的细胞模式。该技术能够在工程组织中产生细胞界面。将用于在表达 PCP 的细胞和表达 Ret 的细胞之间创建界面。基于荧光的激酶活性产生器和数学模型将用于研究这些该提案的目标 2 将在 3D 中对两种细胞类型进行建模。组织支架并研究界面对所得上皮结构的尺寸和形状的影响。该提议的中心假设是 PCP 表达细胞局部限制 Ret-GDNF 驱动的上皮细胞组织在界面处生长，从而产生确定尺寸和形状的结构。该提案中开发的内容将提高我们对导致 CAKUT 的细胞机制的理解缺陷并创建新工具来在人类疾病的类器官模型中构建明确的组织结构。