Hepatocyte nuclear factor-4α regulates the human organic anion transporter 1 gene in the kidney

2007 ◽  
Vol 292 (6) ◽  
pp. F1819-F1826 ◽  
Author(s):  
Ken Ogasawara ◽  
Tomohiro Terada ◽  
Jun-ichi Asaka ◽  
Toshiya Katsura ◽  
Ken-ichi Inui
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Organic Anion ◽  
Critical Role ◽  
Direct Repeat ◽  
Organic Anion Transporter ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Anion Transporter

Human organic anion transporter 1 (OAT1, SLC22A6), which is localized to the basolateral membranes of renal tubular epithelial cells, plays a critical role in the excretion of anionic compounds. OAT1 is regulated by various pathophysiological conditions, but little is known about the molecular mechanisms regulating the expression of OAT1. In the present study, we investigated the transcriptional regulation of OAT1 and found that hepatocyte nuclear factor (HNF)-4α markedly transactivated the OAT1 promoter. A deletion analysis of the OAT1 promoter suggested that the regions spanning −1191 to −700 base pairs (bp) and −140 to −79 bp were essential for the transactivation by HNF-4α. These regions contained a direct repeat separated by two nucleotides (DR-2), which is one of the consensus sequences binding to HNF-4α, and an inverted repeat separated by eight nucleotides (IR-8), which was recently identified as a novel element for HNF-4α, respectively. An electrophoretic mobility shift assay showed that HNF-4α bound to DR-2 and IR-8 under the conditions of HNF-4α overexpression. Furthermore, under normal conditions, HNF-4α bound to IR-8, and a mutation in IR-8 markedly reduced the OAT1 promoter activity, indicating that HNF-4α regulates the basal transcription of OAT1 via IR-8. This paper reports the first characterization of the human OAT1 promoter and the first gene in the kidney whose promoter activity is regulated by HNF-4α.

The Human Organic Anion Transporter GenesOAT5andOAT7Are Transactivated by Hepatocyte Nuclear Factor-1α (HNF-1α)

2010 ◽  
Vol 78 (6) ◽  
pp. 1079-1087 ◽  
Author(s):  
Kerstin Klein ◽  
Christoph Jüngst ◽  
Jessica Mwinyi ◽  
Bruno Stieger ◽  
Franz Krempler ◽  
...  
Keyword(s):  
Organic Anion ◽  
Organic Anion Transporter ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Anion Transporter ◽  
Hepatocyte Nuclear Factor 1Α

The Human Organic Anion Transporter 2 Gene Is Transactivated by Hepatocyte Nuclear Factor-4α and Suppressed by Bile Acids

2005 ◽  
Vol 67 (5) ◽  
pp. 1629-1638 ◽  
Author(s):  
Katrin Popowski ◽  
Jyrki J. Eloranta ◽  
Michael Saborowski ◽  
Michael Fried ◽  
Peter J. Meier ◽  
...  
Keyword(s):  
Bile Acids ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Anion Transporter ◽  
Hepatocyte Nuclear Factor 4Α

scholarly journals Molecular Mechanisms for Species Differences in Organic Anion Transporter 1, OAT1: Implications for Renal Drug Toxicity

2018 ◽  
Vol 94 (1) ◽  
pp. 689-699 ◽  
Author(s):  
Ling Zou ◽  
Adrian Stecula ◽  
Anshul Gupta ◽  
Bhagwat Prasad ◽  
Huan-Chieh Chien ◽  
...  
Keyword(s):  
Drug Toxicity ◽  
Molecular Mechanisms ◽  
Species Differences ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Anion Transporter

Adaptive responses of renal organic anion transporter 3 (OAT3) during cholestasis

2008 ◽  
Vol 295 (1) ◽  
pp. F247-F252 ◽  
Author(s):  
Jiarong Chen ◽  
Tomohiro Terada ◽  
Ken Ogasawara ◽  
Toshiya Katsura ◽  
Ken-ichi Inui
Keyword(s):  
Bile Acids ◽  
Cholic Acid ◽  
Molecular Mechanisms ◽  
Competitive Inhibition ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Specific Substrate ◽  
Renal Secretion ◽  
Sd Rats ◽  
Anion Transporter

During cholestasis, bile acids are mainly excreted into the urine, but adaptive renal responses to cholestasis, especially molecular mechanisms for renal secretion of bile acids, have not been well understood. Organic anion transporters (OAT1 and OAT3) are responsible for membrane transport of anionic compounds at the renal basolateral membranes. In the present study, we investigated the pathophysiological roles of OAT1 and OAT3 in terms of renal handling of bile acids. The Eisai hyperbilirubinemic rats (EHBR), mutant rats without multidrug resistance-associated protein 2, showed higher serum and urinary concentrations of bile acids, compared with Sprague-Dawley (SD) rats (wild type). The protein expression level of rat OAT3 was significantly increased in EHBR compared with SD rats, whereas the expression of rat OAT1 was unchanged. The transport activities of rat and human OAT3, but not OAT1, were markedly inhibited by various bile acids such as chenodeoxycholic acid and cholic acid. Cholic acid, glycocholic acid, and taurocholic acid, which mainly increased during cholestasis, are transported by OAT3. The plasma concentration of β-lactam antibiotic cefotiam, a specific substrate for OAT3, was more increased in EHBR than in SD rats despite upregulation of OAT3 protein. This may be due to the competitive inhibition of cefotiam transport by bile acids via OAT3. In conclusion, the present study clearly demonstrated that OAT3 is responsible for renal secretion of bile acids during cholestasis and that the pharmacokinetic profile of OAT3 substrates may be affected by cholestasis.

scholarly journals Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

2018 ◽  
Vol 314 (1) ◽  
pp. G14-G21 ◽  
Author(s):  
Saminathan Muthusamy ◽  
Jong Jin Jeong ◽  
Ming Cheng ◽  
Jessica A. Bonzo ◽  
Anoop Kumar ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Core Promoter ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Protein Levels ◽  
Hepatocyte Nuclear Factor 4Α

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

scholarly journals Phosphorylation of Ser158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4α transcriptional activity

2006 ◽  
Vol 394 (2) ◽  
pp. 379-387 ◽  
Author(s):  
Hongtao Guo ◽  
Chengjiang Gao ◽  
Zhiyong Mi ◽  
Philip Y. Wai ◽  
Paul C. Kuo
Keyword(s):  
Dna Binding ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
P38 Kinase ◽  
Mobility Shift ◽  
Transactivation Potential ◽  
Hepatocyte Nuclear Factor 4Α

In IL-1β (interleukin 1β)-stimulated rat hepatocytes exposed to superoxide, we have previously identified an IRX (inflammatory redox)-sensitive DR1 [direct repeat of RG(G/T)TCA with one base spacing] cis-acting activator element (nt –1327 to –1315) in the iNOS (inducible nitric oxide synthase) promoter: AGGTCAGGGGACA. The corresponding transcription factor was identified to be HNF4α (hepatocyte nuclear factor-4α). HNF4α DNA binding activity and transactivation potential are tightly regulated by its state of phosphorylation. However, the functional consequences of IRX-mediated post-translational phosphorylation of HNF4α have not been well characterized. In the setting of IL-1β+H2O2, HNF4α functional activity is associated with a unique serine/threonine phosphorylation pattern. This indicates that an IRX-sensitive serine/threonine kinase pathway targets HNF4α to augment hepatocyte iNOS transcription. In the present study, following identification of phosphorylated residues in HNF4α, serial mutations were performed to render the target residues phosphorylation-resistant. Electrophoretic mobility-shift assays and transient transfection studies utilizing the iNOS promoter showed that the S158A mutation ablates IRX-mediated HNF4α DNA binding and transactivation. Gain-of-function mutation with the S158D phosphomimetic HNF4α vector supports a critical role for Ser158 phosphorylation. In vitro phosphorylation and kinase inhibitor studies implicate p38 kinase activity. Our results indicate that p38 kinase-mediated Ser158 phosphorylation is essential for augmentation of the DNA binding and transactivation potential of HNF4α in the presence of IL-1β+H2O2. This pathway results in enhanced iNOS expression in hepatocytes exposed to pro-inflammatory cytokines and oxidative stress.

scholarly journals Renal secretion of hydrochlorothiazide involves organic anion transporter 1/3, organic cation transporter 2, and multidrug and toxin extrusion protein 2-K

2019 ◽  
Vol 317 (4) ◽  
pp. F805-F814
Author(s):  
Jia Yin ◽  
David J. Wagner ◽  
Bhagwat Prasad ◽  
Nina Isoherranen ◽  
Kenneth E. Thummel ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Organic Cation ◽  
Organic Anion ◽  
Organic Cation Transporter ◽  
Tubular Secretion ◽  
Organic Anion Transporter ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Organic Cation Transporter 2 ◽  
Toxin Extrusion

Hydrochlorothiazide (HCTZ) is the most widely used thiazide diuretic for the treatment of hypertension either alone or in combination with other antihypertensives. HCTZ is mainly cleared by the kidney via tubular secretion, but the underlying molecular mechanisms are unclear. Using cells stably expressing major renal organic anion and cation transporters [human organic anion transporter 1 (hOAT1), human organic anion transporter 3 (hOAT3), human organic cation transporter 2 (hOCT2), human multidrug and toxin extrusion 1 (hMATE1), and human multidrug and toxin extrusion 2-K (hMATE2-K)], we found that HCTZ interacted with both organic cation and anion transporters. Uptake experiments further showed that HCTZ is transported by hOAT1, hOAT3, hOCT2, and hMATE2-K but not by hMATE1. Detailed kinetic analysis coupled with quantification of membrane transporter proteins by targeted proteomics revealed that HCTZ is an excellent substrate for hOAT1 and hOAT3. The apparent affinities ( Km) for hOAT1 and hOAT3 were 112 ± 8 and 134 ± 13 μM, respectively, and the calculated turnover numbers ( kcat) were 2.48 and 0.79 s−1, respectively. On the other hand, hOCT2 and hMATE2-K showed much lower affinity for HCTZ. The calculated transport efficiency ( kcat/ Km) at the single transporter level followed the rank order of hOAT1> hOAT3 > hOCT2 and hMATE2-K, suggesting a major role of organic anion transporters in tubular secretion of HCTZ. In vitro inhibition experiments further suggested that HCTZ is not a clinically relevant inhibitor for hOAT1 or hOAT3. However, strong in vivo inhibitors of hOAT1/3 may alter renal secretion of HCTZ. Together, our study elucidated the molecular mechanisms underlying renal handling of HCTZ and revealed potential pathways involved in the disposition and drug-drug interactions for this important antihypertensive drug in the kidney.

scholarly journals Cytotoxicity of Antiviral Nucleotides Adefovir and Cidofovir Is Induced by the Expression of Human Renal Organic Anion Transporter 1

2000 ◽  
Vol 11 (3) ◽  
pp. 383-393 ◽  
Author(s):  
EDMUND S. HO ◽  
DEBORAH C. LIN ◽  
DIRK B. MENDEL ◽  
TOMAS CIHLAR
Keyword(s):  
Organic Anion ◽  
Critical Role ◽  
Antiviral Agents ◽  
Chinese Hamster ◽  
Organic Anion Transporter ◽  
Nucleotide Analogs ◽  
Anion Transporter ◽  
Organ Specific

Abstract. The transport of organic anions in proximal convoluted tubules plays an essential role in the active secretion of a variety of small molecules by the kidney. In addition to other anionic substrates, the human renal organic anion transporter 1 (hOAT1) is capable of transporting the nucleotide analogs adefovir and cidofovir. To investigate the involvement of hOAT1 in the mechanism of nephrotoxicity associated with these two clinically important antiviral agents, Chinese hamster ovary (CHO) cells were stably transfected with hOAT1 cDNA. The resulting CHOhOAT cells showed probenecid-sensitive and pH-dependent uptake of p-aminohippurate (Km = 15.4 μM, Vmax = 20.6 pmol/106 cells · min), a prototypical organic anion substrate. In addition, the stably expressed hOAT1 mediated efficient transport of adefovir (Km = 23.8 μM, Vmax = 46.0 pmol/106 cells · min) and cidofovir (Km = 58.0 μM, Vmax = 103 pmol/106 cells · min) such that the levels of intracellular metabolites of both nucleotides were >100-fold higher in CHOhOAT cells than in parental CHO. Consequently, adefovir and cidofovir were approximately 500-fold and 400-fold more cytotoxic, respectively, in CHOhOAT cells compared to CHO. The cytotoxicity of both drugs in CHOhOAT cells was markedly reduced in the presence of hOAT1 inhibitors. The cyclic prodrug of cidofovir, which exhibits reduced in vivo nephrotoxicity, was a poor substrate for hOAT1 and showed only marginally increased cytotoxicity in CHOhOAT cells. In conclusion, these studies demonstrate that hOAT1 plays a critical role in the organ-specific toxicity of adefovir and cidofovir, and indicates that CHOhOAT cells may represent a useful in vitro model to investigate the potential nephrotoxicity of clinically relevant organic anion agents.

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