UC San Diego

The proximal tubule (PT) is responsible for more than half of the water reabsorption, recovery of organic solutes, and practically all of the clearance of drugs and metabolites within the kidney. Understanding the transcriptional regulation of PT cells is of major biological and clinical importance, yet much remains to be known. This dissertation attempts to address this problem using multiple molecular, cellular and systems biological approaches. Chapter 2 explores the regulation of drug metabolizing enzyme and transporter (collectively referred to as DMEs) expression in the PT, predominantly focusing on the role of hepatocyte nuclear factor 4a (Hnf4a). Systems analysis revealed hepatocyte nuclear factors Hnf1a and Hnf4a as being potential regulators of DME expression in the PT. Examining genomic localization of Hnf4a and enhancer-associated protein E1A binding protein p300 (p300) by ChIP-sequencing provided additional evidence that Hnf1a and Hnf4a are candidate lineage-determining transcription factors for the proximal tubule cellular identity. A small molecule Hnf4a antagonist was used to show that Hnf4a is required for the expression of multiple DMEs in the kidney proximal tubule in an ex vivo kidney organ culture model. Finally, ectopic expression of Hnf1a and Hnf4a in mouse embryonic fibroblasts (MEFs) was used to show that Hnf1a and Hnf4a can induce the transcription of a number of important DMEs, and established functional organic anion transport capacity – a specific property of the PT. Chapter 3 further explores the capacity of Hnf1a and Hnf4a to establish a PT cell-like identity. We show that while transducing MEFs with Hnf1a and Hnf4a does not completely transdifferentiate them to PT-like cells, they do form tight-junctions in culture and express a broad array of transporters and junctional components, both important characteristics of PT cells. We then show that three hepatocyte lineage-determining factors Gata4, Foxa2 and Foxa3 repress the induction of PT signature genes by Hnf1a and Hnf4a, and in turn upregulate the expression of hepatocyte signature genes, thus identifying a transcriptional switch that appears to help confer tissue-specific roles of Hnf1a and Hnf4a.