The Role of Hepatocyte Nuclear Factors 1a and 4a (Hnf1a and Hnf4a) in the Specification and Transcriptional Regulation of the Kidney Proximal Tubule

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

Kidney versus Liver Specification of SLC and ABC Drug Transporters, Tight Junction Molecules, and Biomarkers

The hepatocyte nuclear factors, Hnf1a and Hnf4a, in addition to playing key roles in determining hepatocyte fate, have been implicated as candidate lineage-determining transcription factors in the kidney proximal tubule (PT) [Martovetsky et. al., (2012) Mol Pharmacol 84:808], implying an additional level of regulation that is potentially important in developmental and/or tissue-engineering contexts. Mouse embryonic fibroblasts (MEFs) transduced with Hnf1a and Hnf4a form tight junctions and express multiple PT drug transporters (e.g., Slc22a6/Oat1, Slc47a1/Mate1, Slc22a12/Urat1, Abcg2/Bcrp, Abcc2/Mrp2, Abcc4/Mrp4), nutrient transporters (e.g., Slc34a1/NaPi-2, Slco1a6), and tight junction proteins (occludin, claudin 6, ZO-1/Tjp1, ZO-2/Tjp2). In contrast, the coexpression (with Hnf1a and Hnf4a) of GATA binding protein 4 (Gata4), as well as the forkhead box transcription factors, Foxa2 and Foxa3, in MEFs not only downregulates PT markers but also leads to upregulation of several hepatocyte markers, including albumin, apolipoprotein, and transferrin. A similar result was obtained with primary mouse PT cells. Thus, the presence of Gata4 and Foxa2/Foxa3 appears to alter the effect of Hnf1a and Hnf4a by an as-yet unidentified mechanism, leading toward the generation of more hepatocyte-like cells as opposed to cells exhibiting PT characteristics. The different roles of Hnf4a in the kidney and liver was further supported by reanalysis of ChIP-seq data, which revealed Hnf4a colocalization in the kidney near PT-enriched genes compared with those genes enriched in the liver. These findings provide valuable insight, not only into the developmental, and perhaps organotypic, regulation of drug transporters, drug-metabolizing enzymes, and tight junctions, but also for regenerative medicine strategies aimed at restoring the function of the liver and/or kidney (acute kidney injury, AKI; chronic kidney disease, CKD)

Organic anion and cation SLC22 "drug" transporter (Oat1, Oat3, and Oct1) regulation during development and maturation of the kidney proximal tubule.

Proper physiological function in the pre- and post-natal proximal tubule of the kidney depends upon the acquisition of selective permeability, apical-basolateral epithelial polarity and the expression of key transporters, including those involved in metabolite, toxin and drug handling. Particularly important are the SLC22 family of transporters, including the organic anion transporters Oat1 (originally identified as NKT) and Oat3 as well as the organic cation transporter Oct1. In ex vivo cultures of metanephric mesenchyme (MM; the embryonic progenitor tissue of the nephron) Oat function was evident before completion of nephron segmentation and corresponded with the maturation of tight junctions as measured biochemically by detergent extractability of the tight junction protein, ZO-1. Examination of available time series microarray data sets in the context of development and differentiation of the proximal tubule (derived from both in vivo and in vitro/ex vivo developing nephrons) allowed for correlation of gene expression data to biochemically and functionally defined states of development. This bioinformatic analysis yielded a network of genes with connectivity biased toward Hnf4α (but including Hnf1α, hyaluronic acid-CD44, and notch pathways). Intriguingly, the Oat1 and Oat3 genes were found to have strong temporal co-expression with Hnf4α in the cultured MM supporting the notion of some connection between the transporters and this transcription factor. Taken together with the ChIP-qPCR finding that Hnf4α occupies Oat1, Oat3, and Oct1 proximal promoters in the in vivo differentiating rat kidney, the data suggest a network of genes with Hnf4α at its center plays a role in regulating the terminal differentiation and capacity for drug and toxin handling by the nascent proximal tubule of the kidney

Development of epithelial characteristics and nephron segmentation.

<p>(A) A diagram showing the morphological progression of nephron development ex vivo, and the corresponding culture time. B) Immunofluorescent image of a 2 day (early) nephron culture. The tight junction marker ZO-1 is shown in red, and the adherens junction and marker E-cadherin is shown in green (panels B-D). C) Immunofluorescent image of 3 day (early) nephron culture. D) Immunofluorescent image of the convoluted late tubule (5 days and beyond). E) Immunofluorescent image of a 5 day (late) nephron culture. Peanut lectin marks developing podocytes (red), DAPI (blue). The late nephron begins segment specific differentiation. Bar is 50 µm (B, C, E) or 100 µm (D).</p

Expression and protein localization of Oat1, Oat3, Oct1, and Hnf4α in the proximal tubule.

<p>Evidence from existing literature validates the result of microarray analysis. The genes and protein products of Hnf4α, Oat1, Oat3, and Oct1 are present in the proximal tubule during organogenesis and adulthood.</p

Hepatocyte nuclear factors are associated with Slc22 genes in silico and in vitro.

<p>Evidence from the literature demonstrates the potential for functional regulation of the SLC22 family of genes by hepatocyte nuclear factors.</p

ChIP-qPCR confirms Hnf4α binds the proximal promoters of transporters in the in vivo maturing nephron.

<p>A) ChIP qPCR of Oct1 (Slc22a1), Oat1 (Slc22a6) and Oat3 (Slc22a8) promoters using an HNF4α antibody. Two loci outside of annotated coding or transcribed regions on chromosome 1 and 4 were used as negative controls. B) Schematic workflow of the chromatin immunoprecipitation qPCR experiment.</p

Informatic analysis of cultured nephrons highlights global stages of nephron development.

<p>A) self organizing map (SOM) featuring meta-meta representations of four points in nephron culture. Increasing distance between the samples on the map corresponds to increasing difference in the abstracted transcriptome. B) Non-negative matrix factorization (NMF) of the samples represented in panel A. The arrow highlights metagene F1, which comprises genes highly expressed only after 5 days (120 hours) in culture.</p