Gene regulatory networks in the proximal tubules of the mammalian kidney

项目总结/摘要 肾单位节段由特定类型的上皮细胞组成，这些上皮细胞执行不同的生理功能， 功能并共同作为血液过滤单元。沿着近-远轴，肾小体 其次是近端小管（PT）、Henle袢和远端小管。为了成功改造肾脏 为了替代或刺激肾单位再生，必须了解（1） 肾单位的不同节段在发育过程中是特定的，以及（2）它们的节段身份是如何确定的。 维持在成人肾脏中。我们建议使用Hnf 4a，一种在PT中特异表达的转录因子， 细胞，作为研究哺乳动物肾单位分割的切入点。PT的主要功能之一是 从滤液中重吸收水和重要的小分子。PT功能缺陷导致Fanconi 肾小管综合征（FRTS），以多饮、多尿、糖尿和肾功能丧失为特征。 HNF 4A基因导致人类FRTS。我们发现，在小鼠中，Hnf 4a特异性地在肾单位中缺失， 一个家族引起PT特异性基因的大量下调，PT发育的中断，以及FRTS样突变。 表型这些结果有力地表明，Hnf 4a在PT发展中起着关键作用。hnf 4a是最 在小鼠肾脏的成年PT细胞中高度表达的转录因子。我们分析了公开的 小鼠成年肾脏中的PT特异性ATAC-seq数据显示，在开放性肾脏中最高度富集的DNA基序是 PT细胞中的染色质结构域是Hnf 4a基序。综上所述，这些结果表明，Hnf 4a可能有助于 作为成年PT细胞的主调节器。为了更好地了解Hnf 4a在成人PT细胞中的功能， 我们已经绘制了小鼠成年PT细胞中Hnf 4a的全基因组结合。初步数据显示， Hnf 4a直接与脂肪酸氧化（FAO）相关基因以及编码溶质的SLC基因结合 载体蛋白这两组基因对PT细胞的主要功能--重吸收至关重要。更好地 为了了解Hnf 4a在肾发生过程中如何调节PT细胞的特化，我们将确定Hnf 4a在PT细胞中的作用。 Hnf 4a在PT发展的早期阶段的表达，并鉴定Hnf 4a的直接靶基因。为了研究Hnf 4a是否 Hnf 4a在成人PT细胞的维持/功能中起作用，我们将测试Hnf 4a是否调节FAO的表达。 和SLC基因。我们还将检测Hnf 4a是否是维持开放染色质所必需的 成人PT细胞中的区域。我们提出的研究将填补分子生物学领域长期存在的知识空白。 哺乳动物肾单位PT细胞的特化和维持的潜在机制， 提高我们对FRTS致病机制的理解。

Project Summary/Abstract Nephron segments are composed of specific types of epithelial cells that perform distinct physiological functions and collectively act as a blood filtration unit. Along the proximo-distal axis, the renal corpuscle is followed by the proximal tubule (PT), loop of Henle, and distal tubule. To successfully engineer kidney cells/tissue for replacement or to stimulate nephron regeneration, it is essential to understand (1) how the different segments of the nephron are specified during development and (2) how their segmental identities are maintained in the adult kidney. We propose to use Hnf4a, a transcription factor specifically expressed in PT cells, as an entry point to study mammalian nephron segmentation. One of the major functions of the PT is reabsorption of water and vital small molecules from the filtrate. Defective PT function causes Fanconi renotubular syndrome (FRTS), characterized by polydipsia, polyuria, and glucosuria and loss-of-function of HNF4A gene causes FRTS in humans. We found that, in mice, the deletion of Hnf4a specifically in the nephron lineage caused massive downregulation of PT-specific genes, disruption in PT development, and an FRTS-like phenotype. These results strongly suggest that Hnf4a plays a key role in PT development. Hnf4a is the most highly expressed transcription factor in adult PT cells in the mouse kidney. Our analysis of publicly available PT-specific ATAC-seq data in the mouse adult kidney shows that the most highly enriched DNA motif in open chromatin domains in PT cells is the Hnf4a motif. Taken together, these results suggest that Hnf4a may serve as a master regulator in adult PT cells. In order to better understand the functions of Hnf4a in adult PT cells, we have mapped genome-wide binding of Hnf4a in mouse adult PT cells. Our preliminary data show that Hnf4a directly binds to genes involved in fatty acid oxidation (FAO) and also SLC genes encoding solute carrier proteins. These two sets of genes are critical for reabsorption, the major function of PT cells. To better understand how Hnf4a regulates the specification of PT cells during nephrogenesis, we will determine the role of Hnf4a in early stages of PT development and identify direct target genes of Hnf4a. To investigate if Hnf4a plays a role in the maintenance/function of adult PT cells, we will test if Hnf4a regulates the expression of FAO and SLC genes in adult PT cells. We will also test if Hnf4a is required for the maintenance of open chromatin regions in adult PT cells. Our proposed studies will fill a longstanding gap of knowledge in the molecular mechanisms underlying specification and maintenance of PT cells of the mammalian nephron and significantly improve our understanding of the pathogenic mechanisms of FRTS.