Hepatocyte nuclear factors 4? and 1? regulate kidney developmental expression of drug-metabolizing enzymes and drug transporters.
ABSTRACT: The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule (PT) is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors, which are thought to "sense" deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including hepatocyte nuclear factor 4? (Hnf4a) and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), peroxisome proliferator-activated receptor ? (Ppar?), and Tp53. Motif analysis of cis-regulatory enhancers further suggested that Hnf4a and Hnf1a are the main transcriptional regulators of DMEs in the PT. Available expression data from tissue-specific Hnf4a knockout tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. Chromatin immunoprecipitation combined with massively parallel DNA sequencing was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1 (Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8 (Oat3), and Slc47a1 (Mate1). Conversely, overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts, sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-enriched brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT-like cellular identity. We discuss our data in the context of the "remote sensing and signaling hypothesis" (Ahn and Nigam, 2009; Wu et al., 2011).
Project description:The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors which are thought to “sense” deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including Hnf4a and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), Ppara, and Tp53. Motif analysis of cis-regulatory further suggested that Hnf4a and Hnf1a are the main transcriptional regulators in the PT. Available expression data from tissue-specific Hnf4a KO tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. ChIP-seq was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1(Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8(Oat3), and Slc47a1(Mate1). Conversely, overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts (MEFs), sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-specific brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT cellular identity. Hnf4a binding was examined in rat kidneys at three timepoints (E20, P13 and Adult) and p300 binding was examined in adult rat kidney cortex tissue using ChIP-seq. Four corresponding input DNA samples were used as controls for peak calling.
Project description: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).
Project description:Hepatocyte nuclear factor 4 alpha (HNF4a) is a liver-enriched master regulator of liver function. HNF4a is important in regulating hepatic expression of certain cytochrome P450s. The purpose of this study was to use mice lacking HNF4a expression in liver (HNF4a-HNull) to elucidate the role of HNF4a in regulating hepatic expression of phase II enzymes and transporters in mice. Compared with male wild-type mice, HNF4a-HNull male mouse livers had (1) markedly lower messenger RNAs (mRNAs) encoding the uptake transporters sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide (Oatp) 1a1, Oatp2b1, organic anion transporter 2, sodium phosphate cotransporter type 1, sulfate anion transporter 1, sodium-dependent vitamin C transporter 1, the phase II enzymes Uridine 5'-diphospho (UDP)-glucuronosyltransferase (Ugt) 2a3, Ugt2b1, Ugt3a1, Ugt3a2, sulfotransferase (Sult) 1a1, Sult1b1, Sult5a1, the efflux transporters multidrug resistance-associated protein (Mrp) 6, and multidrug and toxin extrusion 1; (2) moderately lower mRNAs encoding Oatp1b2, organic cation transporter (Oct) 1, Ugt1a5, Ugt1a9, glutathione S-transferase (Gst) m4, Gstm6, and breast cancer resistance protein; but (3) higher mRNAs encoding Oatp1a4, Octn2, Ugt1a1, Sult1e1, Sult2a2, Gsta4, Gstm1-m3, multidrug resistance protein (Mdr) 1a, Mrp3, and Mrp4. Hepatic signaling of nuclear factor E2-related factor 2 and pregnane X receptor appear to be activated in HNF4a-HNull mice. In conclusion, HNF4a deficiency markedly alters hepatic mRNA expression of a large number of phase II enzymes and transporters, probably because of the loss of HNF4a, which is a transactivator and a determinant of gender-specific expression and/or adaptive activation of signaling pathways important in hepatic regulation of these phase II enzymes and transporters.
Project description:Impairment of drug disposition in the liver during inflammation has been attributed to downregulation of gene expression of drug-metabolizing enzymes (DMEs) and drug transporters. Inflammatory responses in the liver are primarily mediated by Toll-like receptors (TLRs). We have recently shown that activation of TLR2 or TLR4 by lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, leads to the downregulation of gene expression of DMEs/transporters. However, the molecular mechanism underlying this downregulation is not fully understood. The xenobiotic nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), regulate the expression of DMEs/transporter genes. Downregulation of DMEs/transporters by LTA or LPS was associated with reduced expression of PXR and CAR genes. To determine the role of CAR, we injected CAR(+/+) and CAR(-/-) mice with LTA or LPS, which significantly downregulated (~40%-60%) RNA levels of the DMEs, cytochrome P450 (Cyp)3a11, Cyp2a4, Cyp2b10, uridine diphosphate glucuronosyltransferase 1a1, amine N-sulfotransferase, and the transporter, multidrug resistance-associated protein 2, in CAR(+/+) mice. Suppression of most of these genes was attenuated in LTA-treated CAR(-/-) mice. In contrast, LPS-mediated downregulation of these genes was not attenuated in CAR(-/-) mice. Induction of these genes by mouse CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene was sustained in LTA- but not in LPS-treated mice. Similar observations were obtained in humanized CAR mice. We have replicated these results in primary hepatocytes as well. Thus, LPS can downregulate DME/transporter genes in the absence of CAR, whereas the effect of LTA on these genes is attenuated in the absence of CAR, indicating the potential involvement of CAR in LTA-mediated downregulation of DME/transporter genes.
Project description:INTRODUCTION:Sandwich-cultured human hepatocytes (SCHHs) are the most common in vitro hepatocyte model used for studying hepatic drug disposition and hepatotoxicity. Targeted quantification of key DME and transporter protein expression is useful for in vitro-in vivo extrapolation of drug and xenobiotic clearance and developing corresponding PBPK models. However, established methods for comprehensive quantification of drug metabolizing enzyme (DMEs) and transporter expression in SCHHs are lacking. In this study, a targeted quantitative proteomic isotope dilution nanoLC-MS/MS method developed in our laboratory was adapted to quantify a panel of phase I & II DMEs and transporter proteins in SCHHs under basal and induced conditions. METHODS:SCHHs were treated with known inducers of DMEs (Rifampin: PXR activator, CITCO: CAR activator) and transporters (CDCA: FXR activator) or with vehicle control (DMSO) for 72?h. Membrane protein was isolated from the SCHHs using a membrane extraction kit and 30??g membrane protein was digested with trypsin. The resulting peptides were analyzed by isotope dilution nanoLC-MS/MS to quantify the DMEs and transporters. RESULTS:Using the method, we could quantify fourteen phase I and ten phase II DMEs, and twelve uptake/efflux transporters, under basal and induced conditions in the SCHHs. Analysis showed donor to donor variation in basal protein levels of CYP450s, UGTs and transporters, and that basal protein expression of CYP450s and UGTs was higher than that of transporters. In addition, induction of key proteins in response to rifampin, CITCO and CDCA was observed. DISCUSSION:We have successfully quantified protein abundance of multiple phase I and II DMEs and uptake and efflux transporters in SCHHs using a method previously developed in our laboratory. Our method is sufficiently sensitive to quantify inter-donor differences in protein concentrations at the basal level as well as changes in protein expression in response to endogenous and exogenous stimuli.
Project description:<h4>Background</h4>There is a significant clinical overlap between patients with hepatocyte nuclear factor (HNF)-1A and HNF4A maturity-onset diabetes of the young (MODY), two forms of monogenic diabetes. HNF1A and HNF4A are transcription factors that control common and partly overlapping sets of target genes. We have previously shown that elevated serum pancreatic stone protein / regenerating protein A (PSP/reg1A) levels can be detected in subjects with HNF1A-MODY. In this study, we investigated whether PSP/reg is differentially regulated by HNF1A and HNF4A.<h4>Methods</h4>Quantitative real-time PCR (qPCR) and Western blotting were used to validate gene and protein expression in cellular models of HNF1A- and HNF4A-MODY. Serum PSP/reg1A levels and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY subjects. The two groups were matched for age, body mass index, diabetes duration, blood pressure, lipid profile and aspirin and statin use.<h4>Results</h4>Inducible repression of HNF1A and HNF4A function in INS-1 cells suggested that PSP/reg induction required HNF4A, but not HNF1A. In contrast, crp gene expression was significantly reduced by repression of HNF1A, but not HNF4A function. PSP/reg levels were significantly lower in HNF4A subjects when compared to HNF1A subjects [9.25 (7.85-12.85) ng/ml vs. 12.5 (10.61-17.87) ng/ml, U-test P?=?0.025]. hsCRP levels were significantly lower in HNF1A-MODY [0.22 (0.17-0.35) mg/L] compared to HNF4A-MODY group [0.81 (0.38-1.41) mg/L, U-test P?=?0.002], Parallel measurements of serum PSP/reg1A and hsCRP levels were able to discriminate HNF1A- and HNF4A-MODY subjects.<h4>Conclusion</h4>Our study demonstrates that two distinct target genes, PSP/reg and crp, are differentially regulated by HNF1A and HNF4A, and provides clinical proof-of-concept that serum PSP/reg1A and hsCRP levels may distinguish HNF1A-MODY from HNF4A-MODY subjects.
Project description:Multispecific drug transporters of the solute carrier and ATP-binding cassette families are highly conserved through evolution, but their true physiologic role remains unclear. Analyses of the organic anion transporter 3 (OAT3; encoded by Slc22a8/Oat3, originally Roct) knockout mouse have confirmed its critical role in the renal handling of common drugs (e.g., antibiotics, antivirals, diuretics) and toxins. Previous targeted metabolomics of the knockout of the closely related Oat1 have demonstrated a central metabolic role, but the same approach with Oat3 failed to reveal a similar set of endogenous substrates. Nevertheless, the Oat3 knockout is the only Oat described so far with a physiologically significant phenotype, suggesting the disturbance of metabolic or signaling pathways. Here we analyzed global gene expression in Oat3 knockout tissue, which implicated OAT3 in phase I and phase II metabolism (drug metabolizing enzymes or DMEs), as well as signaling pathways. Metabolic reconstruction with the recently developed "mouse Recon1" supported the involvement of Oat3 in the aforementioned pathways. Untargeted metabolomics were used to determine whether the predicted metabolic alterations could be confirmed. Many significant changes were observed; several metabolites were tested for direct interaction with mOAT3, whereas others were supported by published data. Oat3 thus appears critical for the handling of phase I (hydroxylation) and phase II (glucuronidation) metabolites. Oat3 also plays a role in bioenergetic pathways (e.g., the tricarboxylic acid cycle), as well as those involving vitamins (e.g., folate), steroids, prostaglandins, gut microbiome products, uremic toxins, cyclic nucleotides, amino acids, glycans, and possibly hyaluronic acid. The data seemingly consistent with the Remote Sensing and Signaling Hypothesis (Ahn and Nigam, 2009; Wu et al., 2011), also suggests that Oat3 is essential for the handling of dietary flavonoids and antioxidants.
Project description:BACKGROUND:Dominant inactivating mutations in HNF1A and HNF4A have been described to cause hyperinsulinism (HI) before evolving to diabetes. However, information available in the literature regarding the clinical phenotype is limited. OBJECTIVE:To report the prevalence of HNF1A and HNF4A mutations in a large cohort of children with HI, and to describe their genotypes and phenotypes. DESIGN:Retrospective descriptive study. METHODS:Medical records were reviewed to extract clinical information. Mutation analysis was carried out for 8 genes associated with HI (ABCC8, KCNJ11, GLUD1, GCK, HADH, HNF4A, HNF1A, and UCP2). RESULTS:HNF1A and HNF4A mutations were identified in 5.9% (12 out of 204; HNF1A?=?7, HNF4A?=?5) of diazoxide-responsive HI probands. The clinical phenotypes were extremely variable. Two children showed evidence of ketone production during hypoglycemia, a biochemical profile atypical for hyperinsulinism. At the time of analysis, diazoxide was discontinued in 5 children at a median age of 6.8?years. None had developed diabetes mellitus at a median age of 7.0?years. CONCLUSIONS:Given the heterogeneous clinical phenotypes of HNF1A- and HNF4A-HI, all children with transient, diazoxide-responsive HI without clear history of perinatal stress, should be screened for HNF1A and HNF4A mutations as it predicts the clinical course and affects the subsequent management plan.
Project description:<h4>Background</h4> Pharmacogenetics is involved in customizing therapy according to the genetic makeup of an individual, and is applicable for chemotherapy, radiotherapy as well as targeted therapy. Drug metabolizing enzymes (DMEs) involving both phase I, and phase II reactions are widely studied. Our study was involved in whole exome sequencing (WES) of cancer patients, followed by analysis for identifying key variations in DMEs, and associated transporters that have a potential impact on treatment outcome. <h4>Methodology</h4> A total of 181 solid tumor patients at stage >/= III were subjected to WES by the SureSelectXT Human All Exon V6 + UTR library preparation kit, and sequencing in the Illumina NextSeq 550 system. Bioinformatics analysis involved use of GATK pipeline, and the variants were further assessed for population frequency, functional impact with annovar insilico algorithms. Further variant information from significant DMEs, and transporters were extracted and analyzed with PharmGKB to assess level of evidence and infer their impact on the pathways involved in drug response. <h4>Results</h4> The total study cohort of 181 solid tumor patients included 60 males, and 121 females respectively. Among DMEs, deleterious mutation in dihydropyrimidine dehydrogenase (DPYD; rs67376798), solute carrier organic anion transporter family member 1B1 (SLCO1B1*5), and cytochrome P450 2D6 (CYP2D6*10) associated with metabolism of anticancer drugs was detected to be in high frequency of 26%, 21% and 25% respectively. <h4>Conclusion</h4> Our analysis detected variations in both phase I and phase II DMEs, as well as associated transporter genes which has been documented to reduce drug efficacy, as well as cause grade 3 and 4 toxicity. Our study reiterates the significance of pharmacogenomics in stratifying patients for appropriate therapy regimen focused at better treatment outcome and quality of life.
Project description:When the organic anion transporter Oat1 was first identified as NKT (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, Nigam SK. J Biol Chem 272: 6471-6478, 1997), it was argued that it, together with Oct1, may be part of a larger subfamily (now known as SLC22) involved in organic ion and xenobiotic transport. The least studied among SLC22 transporters are the so-called unknown substrate transporters (USTs). Here, five novel genes located in a cluster on mouse chromosome 19, immediately between Slc22a8 (Oat3)/Slc22a6 (Oat1) and Slc22a19 (Oat5), were identified as homologs of human USTs. These genes display preferential expression in liver and kidney, and one gene, AB056422, has several splicing variants with differential tissue expression and embryonic expression. Along with Slc22a6, Slc22a8, and Slc22a19, these Usts define the largest known cluster of mammalian Slc22 genes. Given the established functions of Oats, these genes may also be involved in organic anion transport. Usts have characteristic motifs and share a signature residue in the possible active site of transmembrane domain 7, a conserved, positively charged, amino acid, Arg356, possibly a site for interaction with organic anions. In certain species, Oat1 and Oat3 appeared to be highly conserved, whereas the Ust part of this cluster appeared to undergo repeated species-specific amplification, suggesting strong environmental selection pressure, and perhaps providing an explanation for copy number variation in the human locus. One Ust amplification in mouse appears to be recent. This cluster may be coordinately regulated and under selective pressure in a species-specific manner.