Development of an AT2-deficient proximal tubule cell line for transport studies
Angiotensin II is a major regulatory peptide for proximal tubule Na+ reabsorption acting through two distinct receptor subtypes: AT1 and AT2. Physiological or pathological roles of AT2 have been difficult to unravel because angiotensin II can affect Na+ transport either directly via AT2 on luminal o...
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| Veröffentlicht in: | In vitro cellular & developmental biology. Animal 2007-11, Vol.43 (10), p.352-360 |
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| creator | Woost, Philip G Kolb, Robert J Chang, Chung-Ho Finesilver, Margaret Inagami, Tadashi Hopfer, Ulrich |
| description | Angiotensin II is a major regulatory peptide for proximal tubule Na+ reabsorption acting through two distinct receptor subtypes: AT1 and AT2. Physiological or pathological roles of AT2 have been difficult to unravel because angiotensin II can affect Na+ transport either directly via AT2 on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin–angiotensin systems impart considerable complexity to angiotensin's regulation. A transport-competent, proximal tubule cell model that lacks AT2 is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT2-receptor-deficient mice were bred with an Immortomouse®, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT2-receptor-deficient [AT2 (−/−)], Tag heterozygous [Tag (+/−)] F2 offspring were selected for cell line generation. S1 proximal tubule segments were micro-dissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm2), short-circuit current (Isc; 0.2 μA/cm2), and proximal tubule-specific Na3+ – succinate (ΔIsc=0.8 μA/cm2 at 2 mM succinate) and Na3+ – phosphate cotransport (ΔIsc=3 μA/cm2 at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT2 receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT2-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT1-deficient lines should help explain angiotensin II signaling relevant to Na+ transport. |
| doi_str_mv | 10.1007/s11626-007-9061-1 |
| format | Article |
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Denry</contributor><creatorcontrib>Woost, Philip G ; Kolb, Robert J ; Chang, Chung-Ho ; Finesilver, Margaret ; Inagami, Tadashi ; Hopfer, Ulrich ; Sato, J. Denry</creatorcontrib><description>Angiotensin II is a major regulatory peptide for proximal tubule Na+ reabsorption acting through two distinct receptor subtypes: AT1 and AT2. Physiological or pathological roles of AT2 have been difficult to unravel because angiotensin II can affect Na+ transport either directly via AT2 on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin–angiotensin systems impart considerable complexity to angiotensin's regulation. A transport-competent, proximal tubule cell model that lacks AT2 is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT2-receptor-deficient mice were bred with an Immortomouse®, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT2-receptor-deficient [AT2 (−/−)], Tag heterozygous [Tag (+/−)] F2 offspring were selected for cell line generation. S1 proximal tubule segments were micro-dissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm2), short-circuit current (Isc; 0.2 μA/cm2), and proximal tubule-specific Na3+ – succinate (ΔIsc=0.8 μA/cm2 at 2 mM succinate) and Na3+ – phosphate cotransport (ΔIsc=3 μA/cm2 at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT2 receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT2-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT1-deficient lines should help explain angiotensin II signaling relevant to Na+ transport.</description><identifier>ISSN: 1071-2690</identifier><identifier>EISSN: 1543-706X</identifier><identifier>DOI: 10.1007/s11626-007-9061-1</identifier><identifier>PMID: 17963016</identifier><language>eng</language><publisher>Germany: The Society for In Vitro Biology 2007</publisher><subject>Angiotensin ; Angiotensin II ; Angiotensin II - pharmacology ; Animals ; Antibodies ; Antigens ; AT2 ; Biological Transport - drug effects ; Blotting, Southern ; Breeding ; Cell and Tissue Models ; Cell culture ; Cell culture techniques ; Cell Line ; Cell lines ; Cilia ; Circuits ; Cryopreservation ; Cultured cells ; Electrolyte transport ; Electrolytes ; Electrophysiology ; Epithelial cell line ; Epithelial cells ; Epithelial Cells - cytology ; Epithelial Cells - drug effects ; Epithelial Cells - enzymology ; Epithelium ; Female ; Genotype ; Guanylate cyclase ; Guanylate Cyclase - metabolism ; Immortalization ; Immortomouse ; Immunoblotting ; Immunohistochemistry ; Interstitial cells ; Kidney Tubules, Proximal - cytology ; Kidney Tubules, Proximal - drug effects ; Kidney Tubules, Proximal - enzymology ; Kidney Tubules, Proximal - metabolism ; Light microscopy ; Male ; Mice ; Monolayers ; Offspring ; Optical microscopy ; Plasma membranes ; Proximal tubule ; Proximal tubules ; Reabsorption ; Receptor, Angiotensin, Type 2 - deficiency ; Receptors ; Renin ; Reverse Transcriptase Polymerase Chain Reaction ; Short circuit currents ; Short-circuit current ; Simian virus 40 ; Sodium ; Wheat Germ Agglutinins - metabolism</subject><ispartof>In vitro cellular & developmental biology. Animal, 2007-11, Vol.43 (10), p.352-360</ispartof><rights>The Society for In Vitro Biology 2007</rights><rights>Copyright 2007 Society for in vitro Biology</rights><rights>The Society for In Vitro Biology 2007.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-b335t-767ecadd99a488e391913480a0625d773d1aeffeb9392f31559674797372da4b3</citedby><cites>FETCH-LOGICAL-b335t-767ecadd99a488e391913480a0625d773d1aeffeb9392f31559674797372da4b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://bioone.org/doi/pdf/10.1007/s11626-007-9061-1$$EPDF$$P50$$Gbioone$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/40205833$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>314,776,780,799,26955,27901,27902,52338,57992,58225</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17963016$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Sato, J. Denry</contributor><creatorcontrib>Woost, Philip G</creatorcontrib><creatorcontrib>Kolb, Robert J</creatorcontrib><creatorcontrib>Chang, Chung-Ho</creatorcontrib><creatorcontrib>Finesilver, Margaret</creatorcontrib><creatorcontrib>Inagami, Tadashi</creatorcontrib><creatorcontrib>Hopfer, Ulrich</creatorcontrib><title>Development of an AT2-deficient proximal tubule cell line for transport studies</title><title>In vitro cellular & developmental biology. Animal</title><addtitle>In Vitro Cell Dev Biol Anim</addtitle><description>Angiotensin II is a major regulatory peptide for proximal tubule Na+ reabsorption acting through two distinct receptor subtypes: AT1 and AT2. Physiological or pathological roles of AT2 have been difficult to unravel because angiotensin II can affect Na+ transport either directly via AT2 on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin–angiotensin systems impart considerable complexity to angiotensin's regulation. A transport-competent, proximal tubule cell model that lacks AT2 is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT2-receptor-deficient mice were bred with an Immortomouse®, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT2-receptor-deficient [AT2 (−/−)], Tag heterozygous [Tag (+/−)] F2 offspring were selected for cell line generation. S1 proximal tubule segments were micro-dissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm2), short-circuit current (Isc; 0.2 μA/cm2), and proximal tubule-specific Na3+ – succinate (ΔIsc=0.8 μA/cm2 at 2 mM succinate) and Na3+ – phosphate cotransport (ΔIsc=3 μA/cm2 at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT2 receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT2-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT1-deficient lines should help explain angiotensin II signaling relevant to Na+ transport.</description><subject>Angiotensin</subject><subject>Angiotensin II</subject><subject>Angiotensin II - pharmacology</subject><subject>Animals</subject><subject>Antibodies</subject><subject>Antigens</subject><subject>AT2</subject><subject>Biological Transport - drug effects</subject><subject>Blotting, Southern</subject><subject>Breeding</subject><subject>Cell and Tissue Models</subject><subject>Cell culture</subject><subject>Cell culture techniques</subject><subject>Cell Line</subject><subject>Cell lines</subject><subject>Cilia</subject><subject>Circuits</subject><subject>Cryopreservation</subject><subject>Cultured cells</subject><subject>Electrolyte transport</subject><subject>Electrolytes</subject><subject>Electrophysiology</subject><subject>Epithelial cell line</subject><subject>Epithelial cells</subject><subject>Epithelial Cells - cytology</subject><subject>Epithelial Cells - drug effects</subject><subject>Epithelial Cells - enzymology</subject><subject>Epithelium</subject><subject>Female</subject><subject>Genotype</subject><subject>Guanylate cyclase</subject><subject>Guanylate Cyclase - metabolism</subject><subject>Immortalization</subject><subject>Immortomouse</subject><subject>Immunoblotting</subject><subject>Immunohistochemistry</subject><subject>Interstitial cells</subject><subject>Kidney Tubules, Proximal - cytology</subject><subject>Kidney Tubules, Proximal - drug effects</subject><subject>Kidney Tubules, Proximal - enzymology</subject><subject>Kidney Tubules, Proximal - metabolism</subject><subject>Light microscopy</subject><subject>Male</subject><subject>Mice</subject><subject>Monolayers</subject><subject>Offspring</subject><subject>Optical microscopy</subject><subject>Plasma membranes</subject><subject>Proximal tubule</subject><subject>Proximal tubules</subject><subject>Reabsorption</subject><subject>Receptor, Angiotensin, Type 2 - deficiency</subject><subject>Receptors</subject><subject>Renin</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Short circuit currents</subject><subject>Short-circuit current</subject><subject>Simian virus 40</subject><subject>Sodium</subject><subject>Wheat Germ Agglutinins - metabolism</subject><issn>1071-2690</issn><issn>1543-706X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkctKxDAUhoMo3h_AhRIQ3EVzmiZpluIdBmaj4C6k01Po0GnGpBV9e1M6KLjRbPKT851bfkJOgF8C5_oqAqhMsSSZ4QoYbJF9kLlgmqvX7aS5BpYpw_fIQYxLno4BtUv2QBslOKh9Mr_Fd2z9eoVdT31NXUevnzNWYd0smvFtHfxHs3It7YdyaJEusG1p23RIax9oH1wX1z70NPZD1WA8Iju1ayMeb-5D8nJ_93zzyGbzh6eb6xkrhZA900rjwlWVMS4vChQGDIi84I6rTFZaiwoc1jWWRpisFiClUTrXRgudVS4vxSG5mOqm-d4GjL1dNXGczXXoh2jT0lIoI_4EwYicS6USeP4LXPohdGkJm-lCSSkKGCmYqEXwMQas7Tqk7wmfFrgdTbGTKXaUoykWUs7ZpvJQrrD6ydi4kIDTCVjG3ofveM4zLgsx7nA1xcvG-w7_0fILZuidpA</recordid><startdate>200711</startdate><enddate>200711</enddate><creator>Woost, Philip G</creator><creator>Kolb, Robert J</creator><creator>Chang, Chung-Ho</creator><creator>Finesilver, Margaret</creator><creator>Inagami, Tadashi</creator><creator>Hopfer, Ulrich</creator><general>The Society for In Vitro Biology 2007</general><general>Springer Science + Business Media</general><general>Society for In Vitro Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>4T-</scope><scope>7QL</scope><scope>7T7</scope><scope>7TK</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>7QO</scope><scope>7X8</scope></search><sort><creationdate>200711</creationdate><title>Development of an AT2-deficient proximal tubule cell line for transport studies</title><author>Woost, Philip G ; Kolb, Robert J ; Chang, Chung-Ho ; Finesilver, Margaret ; Inagami, Tadashi ; Hopfer, Ulrich</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-b335t-767ecadd99a488e391913480a0625d773d1aeffeb9392f31559674797372da4b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Angiotensin</topic><topic>Angiotensin II</topic><topic>Angiotensin II - pharmacology</topic><topic>Animals</topic><topic>Antibodies</topic><topic>Antigens</topic><topic>AT2</topic><topic>Biological Transport - drug effects</topic><topic>Blotting, Southern</topic><topic>Breeding</topic><topic>Cell and Tissue Models</topic><topic>Cell culture</topic><topic>Cell culture techniques</topic><topic>Cell Line</topic><topic>Cell lines</topic><topic>Cilia</topic><topic>Circuits</topic><topic>Cryopreservation</topic><topic>Cultured cells</topic><topic>Electrolyte transport</topic><topic>Electrolytes</topic><topic>Electrophysiology</topic><topic>Epithelial cell line</topic><topic>Epithelial cells</topic><topic>Epithelial Cells - cytology</topic><topic>Epithelial Cells - drug effects</topic><topic>Epithelial Cells - enzymology</topic><topic>Epithelium</topic><topic>Female</topic><topic>Genotype</topic><topic>Guanylate cyclase</topic><topic>Guanylate Cyclase - metabolism</topic><topic>Immortalization</topic><topic>Immortomouse</topic><topic>Immunoblotting</topic><topic>Immunohistochemistry</topic><topic>Interstitial cells</topic><topic>Kidney Tubules, Proximal - cytology</topic><topic>Kidney Tubules, Proximal - drug effects</topic><topic>Kidney Tubules, Proximal - enzymology</topic><topic>Kidney Tubules, Proximal - metabolism</topic><topic>Light microscopy</topic><topic>Male</topic><topic>Mice</topic><topic>Monolayers</topic><topic>Offspring</topic><topic>Optical microscopy</topic><topic>Plasma membranes</topic><topic>Proximal tubule</topic><topic>Proximal tubules</topic><topic>Reabsorption</topic><topic>Receptor, Angiotensin, Type 2 - deficiency</topic><topic>Receptors</topic><topic>Renin</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Short circuit currents</topic><topic>Short-circuit current</topic><topic>Simian virus 40</topic><topic>Sodium</topic><topic>Wheat Germ Agglutinins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Woost, Philip G</creatorcontrib><creatorcontrib>Kolb, Robert J</creatorcontrib><creatorcontrib>Chang, Chung-Ho</creatorcontrib><creatorcontrib>Finesilver, Margaret</creatorcontrib><creatorcontrib>Inagami, Tadashi</creatorcontrib><creatorcontrib>Hopfer, Ulrich</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Docstoc</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Neurosciences Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>In vitro cellular & developmental biology. Animal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Woost, Philip G</au><au>Kolb, Robert J</au><au>Chang, Chung-Ho</au><au>Finesilver, Margaret</au><au>Inagami, Tadashi</au><au>Hopfer, Ulrich</au><au>Sato, J. Denry</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of an AT2-deficient proximal tubule cell line for transport studies</atitle><jtitle>In vitro cellular & developmental biology. Animal</jtitle><addtitle>In Vitro Cell Dev Biol Anim</addtitle><date>2007-11</date><risdate>2007</risdate><volume>43</volume><issue>10</issue><spage>352</spage><epage>360</epage><pages>352-360</pages><issn>1071-2690</issn><eissn>1543-706X</eissn><abstract>Angiotensin II is a major regulatory peptide for proximal tubule Na+ reabsorption acting through two distinct receptor subtypes: AT1 and AT2. Physiological or pathological roles of AT2 have been difficult to unravel because angiotensin II can affect Na+ transport either directly via AT2 on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin–angiotensin systems impart considerable complexity to angiotensin's regulation. A transport-competent, proximal tubule cell model that lacks AT2 is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT2-receptor-deficient mice were bred with an Immortomouse®, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT2-receptor-deficient [AT2 (−/−)], Tag heterozygous [Tag (+/−)] F2 offspring were selected for cell line generation. S1 proximal tubule segments were micro-dissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm2), short-circuit current (Isc; 0.2 μA/cm2), and proximal tubule-specific Na3+ – succinate (ΔIsc=0.8 μA/cm2 at 2 mM succinate) and Na3+ – phosphate cotransport (ΔIsc=3 μA/cm2 at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT2 receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT2-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT1-deficient lines should help explain angiotensin II signaling relevant to Na+ transport.</abstract><cop>Germany</cop><pub>The Society for In Vitro Biology 2007</pub><pmid>17963016</pmid><doi>10.1007/s11626-007-9061-1</doi><tpages>9</tpages></addata></record> |
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| ispartof | In vitro cellular & developmental biology. Animal, 2007-11, Vol.43 (10), p.352-360 |
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| subjects | Angiotensin Angiotensin II Angiotensin II - pharmacology Animals Antibodies Antigens AT2 Biological Transport - drug effects Blotting, Southern Breeding Cell and Tissue Models Cell culture Cell culture techniques Cell Line Cell lines Cilia Circuits Cryopreservation Cultured cells Electrolyte transport Electrolytes Electrophysiology Epithelial cell line Epithelial cells Epithelial Cells - cytology Epithelial Cells - drug effects Epithelial Cells - enzymology Epithelium Female Genotype Guanylate cyclase Guanylate Cyclase - metabolism Immortalization Immortomouse Immunoblotting Immunohistochemistry Interstitial cells Kidney Tubules, Proximal - cytology Kidney Tubules, Proximal - drug effects Kidney Tubules, Proximal - enzymology Kidney Tubules, Proximal - metabolism Light microscopy Male Mice Monolayers Offspring Optical microscopy Plasma membranes Proximal tubule Proximal tubules Reabsorption Receptor, Angiotensin, Type 2 - deficiency Receptors Renin Reverse Transcriptase Polymerase Chain Reaction Short circuit currents Short-circuit current Simian virus 40 Sodium Wheat Germ Agglutinins - metabolism |
| title | Development of an AT2-deficient proximal tubule cell line for transport studies |
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