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

Diagn Mol Pathol. 2003 Jun;12(2):108-17. doi: 10.1097/00019606-200306000-00007.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / pharmacology
  • Crotalid Venoms / toxicity
  • Disease Models, Animal
  • Endothelin-1 / genetics*
  • Endothelin-1 / metabolism
  • Gene Expression*
  • Glomerular Mesangium / drug effects
  • Glomerular Mesangium / metabolism
  • Glomerular Mesangium / pathology*
  • Glomerulonephritis, Membranoproliferative / etiology
  • Glomerulonephritis, Membranoproliferative / metabolism
  • Glomerulonephritis, Membranoproliferative / pathology*
  • Laser Therapy
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Microdissection / methods*
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Thy-1 Antigens / immunology


  • Antibodies, Monoclonal
  • Crotalid Venoms
  • Endothelin-1
  • RNA, Messenger
  • Thy-1 Antigens