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
Nephrology, Dialysis and Transplantation
Background. In the glomerular mesangium, immunologic anduor infectious activation of the inflammatory, NF-kB-mediated signal pathway can induce a progression of already existing mesangial lesions in nonimmunologic 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
... 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 glomeruliucase were isolated from 5 mm 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 a-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 (C t ) method, normalized for the housekeeping gene b-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 C t -ratio was formed between b-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 a-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.