Nephrol Dial Transplant (2003) 18: 710-716
© 2003 European Renal Association-European Dialysis and Transplant Association
A novel evaluation method for paraffinized human renal biopsies using quantitative analysis of microdissected glomeruli and VCAM-1 as marker of inflammatory mesangial cell activation
1 Department of Pathology, University of Cologne and 2 Department of Internal Medicine I, Cologne General Hospital, Merheim Medical Center, Cologne, Germany
Background. In the glomerular mesangium, immunologic and/or infectious activation of the inflammatory, NF-
B-mediated signal pathway can induce a progression of already existing mesangial lesions in non-immunologic and immunologic glomerular disease. This progression is preceded by upregulated mesangial gene expression of which the vascular cell adhesion molecule-1, VCAM-1 (vascular cell adhesion molecule-1), is a well-established marker. Its evaluation on minimal tissue such as routinely paraffinized needle core biopsies is not established and needs the development of a novel evaluation method more meaningful than common immunhistology.
Methods. By laser-microdissection, 10 glomeruli/case were isolated from 5 µm thick tissue slices in a total of 15 cases of mesangial proliferation with different renal diseases (IgA nephropathy, lupus nephritis and mesangial proliferative lesions of unknown aetiology) vs transplant biopsies as negative and TNF 
-treated cultured human mesangial cells as positive controls. After reverse transcription of isolated RNA, cDNA aliquots were quantified for VCAM-1 expression by real-time PCR using the threshold cycle (Ct) method, normalized for the housekeeping gene ß-actin, and compared with qualitative RT–PCR results.
Results. Unsuspected VCAM-1 transcript steady-state levels could be detected by real-time PCR in agreement with qualitative PCR, while morphologic and immunhistologic analyses were unrevealing. As yields of RNA extraction in femtogram quantities cannot be measured spectrophotometrically, a Ct-ratio was formed between ß-actin and VCAM-1 per case showing high VCAM-1 expression in lupus nephritis (1.39), and moderate expression in IgA nephropathy (1.08–1.23) vs TNF -treated mesangial cells (0.97–1.23) and negative control cases (0.66–0.68).
Conclusions. This is the first reported gene expression analysis method for routinely paraffininzed human renal biopsies, demonstrating the power of combined laser-microdissection and PCR quantification as novel methods for the evaluation of minimal tissue beyond purely descriptive morphologic analysis.
Keywords: human; kidney; laser-microdissected glomeruli; paraffinined biopsies; quantitative RT–PCR; VCAM-1
Correspondence and offprint requests to: Dr Jochen W. U. Fries, Institut für Pathologie, Universitaet Koeln, Joseph-Stelzmann-Strasse 9, D-50931 Köln, Germany. Email: jochen.fries{at}uni-koeln.de
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Hoftberger, F. Garzuly, H.P. Dienes, J. Grubits, B. Rohonyi, G. Fischer, Z. Hanzely, H. Lassmann, and H. Budka Fulminant central nervous system demyelination associated with interferon-{alpha} therapy and hepatitis C virus infection Multiple Sclerosis, November 1, 2007; 13(9): 1100 - 1106. [Abstract] [PDF]
|
|