Gene regulatory networks in the proximal tubules of the mammalian kidney

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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.