Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis
Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibro...
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| Veröffentlicht in: | Journal of the American Society of Nephrology 2014-09, Vol.25 (9), p.1979-1990 |
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| container_end_page | 1990 |
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| container_issue | 9 |
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| container_title | Journal of the American Society of Nephrology |
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| creator | GRGIC, Ivica KRAUTZBERGER, A. Michaela HUMPHREYS, Benjamin D HOFMEISTER, Andreas LALLI, Matthew DIROCCO, Derek P FLEIG, Susanne V JING LIU DUFFIELD, Jeremy S MCMAHON, Andrew P ARONOW, Bruce |
| description | Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies. |
| doi_str_mv | 10.1681/asn.2013101143 |
| format | Article |
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Michaela ; HUMPHREYS, Benjamin D ; HOFMEISTER, Andreas ; LALLI, Matthew ; DIROCCO, Derek P ; FLEIG, Susanne V ; JING LIU ; DUFFIELD, Jeremy S ; MCMAHON, Andrew P ; ARONOW, Bruce</creator><creatorcontrib>GRGIC, Ivica ; KRAUTZBERGER, A. Michaela ; HUMPHREYS, Benjamin D ; HOFMEISTER, Andreas ; LALLI, Matthew ; DIROCCO, Derek P ; FLEIG, Susanne V ; JING LIU ; DUFFIELD, Jeremy S ; MCMAHON, Andrew P ; ARONOW, Bruce</creatorcontrib><description>Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies.</description><identifier>ISSN: 1046-6673</identifier><identifier>EISSN: 1533-3450</identifier><identifier>DOI: 10.1681/asn.2013101143</identifier><identifier>PMID: 24652793</identifier><identifier>CODEN: JASNEU</identifier><language>eng</language><publisher>Washington, DC: American Society of Nephrology</publisher><subject>Animals ; Basic Research ; Biological and medical sciences ; Collagen Type I - genetics ; Disease Models, Animal ; Fibrosis ; Gene Expression Profiling - methods ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; Kidney - injuries ; Kidney - metabolism ; Kidney - pathology ; Kidneys ; Medical sciences ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mice, Transgenic ; Myofibroblasts - metabolism ; Myofibroblasts - pathology ; Nephrology. Urinary tract diseases ; Oligonucleotide Array Sequence Analysis ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Ribosomal Proteins - genetics ; Ribosomal Proteins - metabolism ; RNA, Messenger - genetics ; RNA, Messenger - metabolism ; Up-Regulation ; Ureteral Obstruction - genetics ; Ureteral Obstruction - metabolism ; Ureteral Obstruction - pathology ; Urinary system involvement in other diseases. Miscellaneous</subject><ispartof>Journal of the American Society of Nephrology, 2014-09, Vol.25 (9), p.1979-1990</ispartof><rights>2015 INIST-CNRS</rights><rights>Copyright © 2014 by the American Society of Nephrology.</rights><rights>Copyright © 2014 by the American Society of Nephrology 2014</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c486t-91ed6d51b478e2dcf253b61496872741c19b1bbd3f6bf64796e44ccc47479d593</citedby><cites>FETCH-LOGICAL-c486t-91ed6d51b478e2dcf253b61496872741c19b1bbd3f6bf64796e44ccc47479d593</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147988/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147988/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=28772293$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24652793$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>GRGIC, Ivica</creatorcontrib><creatorcontrib>KRAUTZBERGER, A. Michaela</creatorcontrib><creatorcontrib>HUMPHREYS, Benjamin D</creatorcontrib><creatorcontrib>HOFMEISTER, Andreas</creatorcontrib><creatorcontrib>LALLI, Matthew</creatorcontrib><creatorcontrib>DIROCCO, Derek P</creatorcontrib><creatorcontrib>FLEIG, Susanne V</creatorcontrib><creatorcontrib>JING LIU</creatorcontrib><creatorcontrib>DUFFIELD, Jeremy S</creatorcontrib><creatorcontrib>MCMAHON, Andrew P</creatorcontrib><creatorcontrib>ARONOW, Bruce</creatorcontrib><title>Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis</title><title>Journal of the American Society of Nephrology</title><addtitle>J Am Soc Nephrol</addtitle><description>Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies.</description><subject>Animals</subject><subject>Basic Research</subject><subject>Biological and medical sciences</subject><subject>Collagen Type I - genetics</subject><subject>Disease Models, Animal</subject><subject>Fibrosis</subject><subject>Gene Expression Profiling - methods</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>Kidney - injuries</subject><subject>Kidney - metabolism</subject><subject>Kidney - pathology</subject><subject>Kidneys</subject><subject>Medical sciences</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Mice, Inbred DBA</subject><subject>Mice, Transgenic</subject><subject>Myofibroblasts - metabolism</subject><subject>Myofibroblasts - pathology</subject><subject>Nephrology. Urinary tract diseases</subject><subject>Oligonucleotide Array Sequence Analysis</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Ribosomal Proteins - genetics</subject><subject>Ribosomal Proteins - metabolism</subject><subject>RNA, Messenger - genetics</subject><subject>RNA, Messenger - metabolism</subject><subject>Up-Regulation</subject><subject>Ureteral Obstruction - genetics</subject><subject>Ureteral Obstruction - metabolism</subject><subject>Ureteral Obstruction - pathology</subject><subject>Urinary system involvement in other diseases. 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Michaela ; HUMPHREYS, Benjamin D ; HOFMEISTER, Andreas ; LALLI, Matthew ; DIROCCO, Derek P ; FLEIG, Susanne V ; JING LIU ; DUFFIELD, Jeremy S ; MCMAHON, Andrew P ; ARONOW, Bruce</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c486t-91ed6d51b478e2dcf253b61496872741c19b1bbd3f6bf64796e44ccc47479d593</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Animals</topic><topic>Basic Research</topic><topic>Biological and medical sciences</topic><topic>Collagen Type I - genetics</topic><topic>Disease Models, Animal</topic><topic>Fibrosis</topic><topic>Gene Expression Profiling - methods</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>Kidney - injuries</topic><topic>Kidney - metabolism</topic><topic>Kidney - pathology</topic><topic>Kidneys</topic><topic>Medical sciences</topic><topic>Mice</topic><topic>Mice, Inbred C57BL</topic><topic>Mice, Inbred DBA</topic><topic>Mice, Transgenic</topic><topic>Myofibroblasts - metabolism</topic><topic>Myofibroblasts - pathology</topic><topic>Nephrology. 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Michaela</au><au>HUMPHREYS, Benjamin D</au><au>HOFMEISTER, Andreas</au><au>LALLI, Matthew</au><au>DIROCCO, Derek P</au><au>FLEIG, Susanne V</au><au>JING LIU</au><au>DUFFIELD, Jeremy S</au><au>MCMAHON, Andrew P</au><au>ARONOW, Bruce</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis</atitle><jtitle>Journal of the American Society of Nephrology</jtitle><addtitle>J Am Soc Nephrol</addtitle><date>2014-09-01</date><risdate>2014</risdate><volume>25</volume><issue>9</issue><spage>1979</spage><epage>1990</epage><pages>1979-1990</pages><issn>1046-6673</issn><eissn>1533-3450</eissn><coden>JASNEU</coden><abstract>Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies.</abstract><cop>Washington, DC</cop><pub>American Society of Nephrology</pub><pmid>24652793</pmid><doi>10.1681/asn.2013101143</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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| issn | 1046-6673 1533-3450 |
| language | eng |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central |
| subjects | Animals Basic Research Biological and medical sciences Collagen Type I - genetics Disease Models, Animal Fibrosis Gene Expression Profiling - methods Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism Kidney - injuries Kidney - metabolism Kidney - pathology Kidneys Medical sciences Mice Mice, Inbred C57BL Mice, Inbred DBA Mice, Transgenic Myofibroblasts - metabolism Myofibroblasts - pathology Nephrology. Urinary tract diseases Oligonucleotide Array Sequence Analysis Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Ribosomal Proteins - genetics Ribosomal Proteins - metabolism RNA, Messenger - genetics RNA, Messenger - metabolism Up-Regulation Ureteral Obstruction - genetics Ureteral Obstruction - metabolism Ureteral Obstruction - pathology Urinary system involvement in other diseases. Miscellaneous |
| title | Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis |
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