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.
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.
Dimmler
A
A
Department of Pathology, University of Erlangen-Nürnberg, Germany.
Haas
C S
CS
Cho
S
S
Hattler
M
M
Forster
C
C
Peters
H
H
Schöcklmann
H O
HO
Amann
K
K
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
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D019332:Endothelin-1; D012333:RNA, Messenger; D018800:Thy-1 Antigens