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.

摘要

BACKGROUND:In the glomerular mesangium, immunologic and/or infectious activation of the inflammatory, NF-kappaB-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 micro m 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 alpha-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 beta-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 beta-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 alpha-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.