Localization and Functional Characterization of the Rat Oatp4c1 Transporter in an In Vitro Cell System and Rat Tissues
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{"title"=>"Localization and Functional Characterization of the Rat Oatp4c1 Transporter in an In Vitro Cell System and Rat Tissues", "type"=>"journal", "authors"=>[{"first_name"=>"Kuei-Ling", "last_name"=>"Kuo"}, {"first_name"=>"Haining", "last_name"=>"Zhu"}, {"first_name"=>"Patrick J.", "last_name"=>"McNamara"}, {"first_name"=>"Markos", "last_name"=>"Leggas"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203", "pmid"=>"22768102", "issn"=>"1932-6203", "doi"=>"10.1371/journal.pone.0039641"}, "id"=>"0a15fd9c-6aca-39ea-bc91-b70aa5894db8", "abstract"=>"The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature.", "link"=>"http://www.mendeley.com/research/localization-functional-characterization-rat-oatp4c1-transporter-vitro-cell-system-rat-tissues", "reader_count"=>20, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>1, "Student > Ph. D. Student"=>3, "Student > Master"=>3, "Other"=>4, "Student > Bachelor"=>6, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>1, "Student > Ph. D. Student"=>3, "Student > Master"=>3, "Other"=>4, "Student > Bachelor"=>6, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Agricultural and Biological Sciences"=>10, "Medicine and Dentistry"=>5, "Business, Management and Accounting"=>1, "Linguistics"=>1, "Computer Science"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>10}, "Computer Science"=>{"Computer Science"=>2}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Linguistics"=>{"Linguistics"=>1}}, "reader_count_by_country"=>{"Mexico"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/320563", "https://ndownloader.figshare.com/files/320604", "https://ndownloader.figshare.com/files/320650", "https://ndownloader.figshare.com/files/320713", "https://ndownloader.figshare.com/files/320730", "https://ndownloader.figshare.com/files/320748"], "description"=>"<div><p>The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our <em>in vitro</em> studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature.</p> </div>", "links"=>[], "tags"=>["localization", "characterization", "oatp4c1", "transporter", "tissues"], "article_id"=>123273, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.s001", "https://dx.doi.org/10.1371/journal.pone.0039641.s002", "https://dx.doi.org/10.1371/journal.pone.0039641.s003", "https://dx.doi.org/10.1371/journal.pone.0039641.s004", "https://dx.doi.org/10.1371/journal.pone.0039641.s005", "https://dx.doi.org/10.1371/journal.pone.0039641.s006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Localization_and_Functional_Characterization_of_the_Rat_Oatp4c1_Transporter_in_an_In_Vitro_Cell_System_and_Rat_Tissues/123273", "title"=>"Localization and Functional Characterization of the Rat Oatp4c1 Transporter in an <em>In Vitro</em> Cell System and Rat Tissues", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-06-29 00:54:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/615144"], "description"=>"<p>(A) Western blot analysis for Oatp4c1 expression. Protein lysates (50 µg per lane) from MDCKII cells transfected with pcDNA (empty vector) and Oatp4c1 were prepared from cells grown in monolayers. (B) Immunohistochemical analysis for Oatp4c1 expression. Paraformaldehyde-fixed paraffin-embedded cell sections were stained with PA1343. Color development with NovaRed signifies Oatp4c1 staining. All sections were counterstained with hemotoxylin. Rabbit IgG was used as a negative control. (C) Immunolocalization of Oatp4c1 in polarized MDCKII cells. Cells were double stained with Oatp4c1 (red) and ZO-1 (green). Nuclei were stained with DAPI (blue). Center image in the Oatp4c1 panel is a single optical section of the x–y plane while top and right images represent x–z and y–z planes, respectively, reconstructed from image stacks. The apical and basal sides can be demarcated by ZO-1 and the nuclei, respectively, in both x–z and y–z sections. (D) Western blot analysis of Oatp4c1 in proteins isolated from either the apical (AP) or basolateral (BL) membranes of polarized MDCKII cells. Surface proteins were isolated by biotin-streptavidin labeling on either the apical AP or BL compartment. Na<sup>+</sup>/K<sup>+</sup>-ATPase served as a BL marker to demonstrate the relative efficiency of AP and BL membrane separation. (Magnification (40×)).</p>", "links"=>[], "tags"=>["oatp4c1", "subcellular", "localization", "mdckii"], "article_id"=>285635, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Validation_of_Oatp4c1_expression_and_subcellular_localization_in_MDCKII_cells_/285635", "title"=>"Validation of Oatp4c1 expression and subcellular localization in MDCKII cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-29 01:33:55"}
  • {"files"=>["https://ndownloader.figshare.com/files/615324"], "description"=>"<p>(A) Western blot analysis for Oatp4c1 expression in rat tissues. Crude membrane fractions from rat liver and kidney were prepared. PNGaseF was used for deglycosylation. Oatp4c1 was detected by PA1343. β-actin was used as a loading control. (B) Immunohistochemical analysis for Oatp4c1 subcellular localization in rat tissues. Paraformaldehyde-fixed paraffin-embedded rat tissue sections were stained with PA1343. Color development with NovaRed signifies Oatp4c1 staining. (C) Double immunofluorescence staining for Oatp4c1 (green) and E-cadherin (red) in rat kidney. E-cadherin served as a basolateral marker (Magnification: 40×).</p>", "links"=>[], "tags"=>["localization"], "article_id"=>285826, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Oatp4c1_expression_and_localization_in_rat_kidney_/285826", "title"=>"Oatp4c1 expression and localization in rat kidney.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-29 01:37:06"}
  • {"files"=>["https://ndownloader.figshare.com/files/615437"], "description"=>"<p>(A) Western blot analysis for Oatp4c1 expression in BBM and BLM. Biochemical separation of membrane proteins (20 µg per lane) from rat kidney cortex was achieved by Percoll gradient. Na<sup>+</sup>/K<sup>+</sup>-ATPase alpha 1 was used as a basolateral marker. Abcg2 and Mrp4 served as apical markers. Oatp4c1 was detected by PA1343. (B) LC-MS/MS based proteomic analysis of ∼60–150 kD proteins isolated from BBM and BLM fractions. Spectral counts detected in protein digests obtained from ∼60–150 kD sections excised from the SDS-PAGE gel. Proteins with 3 or more spectral counts in one fraction are presented as “Apical” or “Basolateral” based on previously published data. Spectral counts indicate the number of unique peptides detected in each fraction. (AP, apical).</p>", "links"=>[], "tags"=>["oatp4c1", "kidney", "membrane", "basolateral"], "article_id"=>285928, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_Oatp4c1_in_isolated_kidney_brush_border_membrane_BBM_and_basolateral_membrane_BLM_fractions_/285928", "title"=>"Expression of Oatp4c1 in isolated kidney brush border membrane (BBM) and basolateral membrane (BLM) fractions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-29 01:38:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/615805"], "description"=>"<p>Paraformaldehyde-fixed paraffin-embedded kidney sections were stained with antibodies as indicated. Following staining with PA1343 antibody, color development with NovaRed signifies Oatp4c1 staining (A). Sections were double-stained with PA1343 (B, C, red) and the proximal tubule marker AQP1 (B, green) or the distal tubule marker calbindin (C, green). Nuclei (blue) were stained with DAPI (Magnification: 4× (A), 10× (B, C)).</p>", "links"=>[], "tags"=>["localization", "paraformaldehyde-fixed", "paraffin-embedded", "kidney"], "article_id"=>286295, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Oatp4c1_localization_in_paraformaldehyde_fixed_paraffin_embedded_rat_kidney_tissue_sections_/286295", "title"=>"Oatp4c1 localization in paraformaldehyde-fixed paraffin-embedded rat kidney tissue sections.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-29 01:44:55"}
  • {"files"=>["https://ndownloader.figshare.com/files/616071"], "description"=>"<p>Paraformaldehyde-fixed paraffin-embedded rat kidney sections were stained for Oatp4c1 (red), Mrp4 (red), P-gp (green), and Abcg2 (red) (A). Sections were double stained for Mrp4 (red) and Oatp4c1 (green); (C) P-gp (green) and Oatp4c1 (red); and (D) Abcg2 (red) and Oatp4c1 (green) (B). Yellow/orange color in merged panels indicates colocalization. Nuclei (blue) were stained with DAPI. Images presented in panel 5A were stitched from six individual images using NIH Image J with Fiji plugin (Magnification: 4× (A), 10× (B–D)).</p>", "links"=>[], "tags"=>["colocalization", "efflux", "transporters", "kidney"], "article_id"=>286565, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Oatp4c1_colocalization_with_major_efflux_transporters_in_the_kidney_cortex_/286565", "title"=>"Oatp4c1 colocalization with major efflux transporters in the kidney cortex.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-29 01:49:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/616212"], "description"=>"<p>Time dependent [<sup>3</sup>H]-E3S uptake at pH 5.5 by MDCKII-pcDNA and MDCKII-Oatp4c1 cells during incubation with 0.5 µM [<sup>3</sup>H]-E3S (A). Time dependent Oatp4c1 mediated uptake of [<sup>3</sup>H]-E3S at pH 4.5, 5.5 and 7.4 (B). Concentration dependent [<sup>3</sup>H]-E3S uptake in MDCKII-pcDNA and MDCKII-Oatp4c1 cells after incubation at pH 5.5 for 1 min (C). Concentration dependent Oatp4c1 mediated uptake (1 min) of [<sup>3</sup>H]-E3S at pH 4.5, 5.5 and 7.4 (D). GF: GF120918, MTX: methotrexate. Each point represents the mean ± S.D. of triplicates. *p<0.05, significant differences from control.</p>", "links"=>[], "tags"=>["oatp4c1-mediated", "e3s"], "article_id"=>286699, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Characteristics_of_Oatp4c1_mediated_E3S_transport_/286699", "title"=>"Characteristics of Oatp4c1-mediated E3S transport.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-29 01:51:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/616321"], "description"=>"<p>The uptake clearance was estimated using the parameter estimates in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039641#pone-0039641-t001\" target=\"_blank\">Table 1</a>. The solid lines show the best fit to the data using obtained from [<sup>3</sup>H]-E3S uptake (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039641#pone-0039641-g006\" target=\"_blank\">Fig. 6</a>).</p>", "links"=>[], "tags"=>["ph", "uptake"], "article_id"=>286816, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_effect_of_pH_on_uptake_transport_clearance_/286816", "title"=>"The effect of pH on uptake transport clearance.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-29 01:53:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/616368"], "description"=>"<p>Inhibition of Oatp4c1-mediated [<sup>3</sup>H]-E3S uptake (1 min) was determined in the absence (control) and presence of various compounds (100 µM) at pH 5.5 (A) and 7.4 (B). Oatp4c1-mediated uptake was estimated after subtracting the nonspecific uptake measured in pcDNA cells treated under identical conditions. In all studies the [<sup>3</sup>H]-E3S concentration was 0.5 µM. GF: GF120918, MTX: methotrexate. Each point represents the mean ± S.D. of triplicates. *p<0.05, significant differences from control.</p>", "links"=>[], "tags"=>["uremic", "toxins", "anionic", "physiological", "substances", "oatp4c1-mediated", "e3s"], "article_id"=>286858, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.g008"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_effect_of_uremic_toxins_and_anionic_physiological_substances_on_Oatp4c1_mediated_E3S_transport_/286858", "title"=>"The effect of uremic toxins and anionic physiological substances on Oatp4c1-mediated E3S transport.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-06-29 01:54:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/616463"], "description"=>"<p>The parameters were estimated by fitting a sigmoid-E<sub>max</sub> model to the data. Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test for all pairwise comparisons. *, **, *** p<0.05 indicate significant differences between pH 4.5 and 5.5, pH 4.5 and 7.4, and pH 5.5 and 7.4, respectively.</p>", "links"=>[], "tags"=>["kinetic", "parameters", "oatp4c1-mediated", "e3s", "ph"], "article_id"=>286952, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology", "Genetics", "Physiology"], "users"=>["Kuei-Ling Kuo", "Haining Zhu", "Patrick J. McNamara", "Markos Leggas"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0039641.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transport_kinetic_parameters_of_Oatp4c1_mediated_E3S_transport_at_pH_4_5_5_5_and_7_4_mean_177_S_E_/286952", "title"=>"Transport kinetic parameters of Oatp4c1-mediated E3S transport at pH 4.5, 5.5 and 7.4 (mean ± S.E.).", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-06-29 01:55:52"}

PMC Usage Stats | Further Information

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