title: Laser capture microdissection and real-time PCR for analysis of glomerular endothelin-1 gene expression in mesangiolysis of rat anti-Thy 1.1 and murine Habu Snake Venom glomerulonephritis.
abstract: Molecular analysis of pathologic changes in glomeruli requires methods allowing rapid and exact detection of alterations in gene expression. Here, we analyzed endothelin-1 (ET-1) mRNA expression in mesangiolytic glomeruli during the course of a rat and murine model of mesangioproliferative glomerulonephritis (GN). A novel method combining laser capture microdissection (LCM), which permits the precise removal of selected mesangiolytic glomeruli, with a highly sensitive real-time RT-PCR technique was used. Anti-Thy 1.1. GN was introduced in male Sprague-Dawley rats (1.0 mg/kg body weight of OX-7 IV) and Habu Snake Venom GN was introduced in C57BL6 mice (habu snake venom toxin 6 mg/kg body weight IV). The degree of mesangiolysis during both GNs was analyzed using a semiquantitative scoring system. Mesangiolytic glomeruli were microdissected at different days of the diseases (day 2, 6, and 12 in anti-Thy 1.1 GN and days 1, 3, 7, and 14 in Habu Snake Venom GN) and from normal control animals. After RNA extraction and cDNA synthesis, ET-1 gene expression was measured by real-time RT-PCR. In parallel, in anti-Thy 1.1. GN ET-1 mRNA expression was analyzed using semiquantitative nonradioactive in situ hybridization; ET-1 protein expression was investigated by immunohistochemistry. Mesangiolysis peaked at day 6 in anti-Thy1.1 GN and at day 1 in Habu Snake Venom GN. Mesangiolytic glomeruli were easily microdissected on cryostat sections in both models; quantification of mRNA with RT-PCR was reliable and reproducible. Glomerular ET-1 mRNA expression increased during the course of anti-Thy 1.1 GN and Habu Snake Venom GN peaked when mesangiolysis was most pronounced. This was seen by RT-PCR after glomerular LCM and by in situ hybridization; in parallel, glomerular ET-1 protein expression was increased. Combination of LCM and RT-PCR is a reliable method for quantification of localized gene expression in isolated renal structures. The above data argue for an important role of ET-1 in pathogenesis and/or repair of mesangiolysis in experimental mesangioproliferative GN.
authors.affiliation: Department of Pathology, University of Erlangen-Nürnberg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Dimmler
authors.forename: C S
authors.initials: CS
authors.lastname: Haas
authors.forename: S
authors.initials: S
authors.lastname: Cho
authors.forename: M
authors.initials: M
authors.lastname: Hattler
authors.forename: C
authors.initials: C
authors.lastname: Forster
authors.forename: H
authors.initials: H
authors.lastname: Peters
authors.forename: H O
authors.initials: HO
authors.lastname: Schöcklmann
authors.forename: K
authors.initials: K
authors.lastname: Amann
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D003435:Crotalid Venoms / Q000633:toxicity; D004195:Disease Models, Animal; D019332:Endothelin-1 / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D015432:Glomerulonephritis, Membranoproliferative / Q000209:etiology / Q000378:metabolism / Q000473:pathology*; D053685:Laser Therapy; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018800:Thy-1 Antigens / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D019332:Endothelin-1; D012333:RNA, Messenger; D018800:Thy-1 Antigens