Compartment-specific quantitative gene expression analysis after laser microdissection from archival renal allograft biopsies.
BACKGROUND
Various immunological and non-immunological pathomechanisms are responsible for the cellular damage in renal allografts. Since the kidney is an anatomically complex organ with functional and morphological heterogeneous compartments (interstitium, tubuli, vessels, glomeruli), the local response to injury maybe variable, therefore, the identification of local pathomechanisms is important.


OBJECTIVE
To elucidate any discrepancies in quantitative mRNA expression profiles between a total specimen analysis and a cell-specific evaluation after laser microdissection.


METHODS
Real-time RT-PCR was performed for complement component C3 and heme oxygenase-1 (HO-1) genes compared to the housekeeping gene beta-actin using whole section RNA extracted from formalin-fixed and paraffin-embedded archival material of 16 explanted, rejected renal allografts. Ten non-transplant nephrectomies served as controls. For five cases from each group, five different compartments of the organs (interstitium, proximal tubuli, distal tubuli, vessels, glomeruli) were microdissected and quantitative analysis for C3 and HO-1 was performed identically.


RESULTS
Whole section mRNA expression analysis: the data showed a constant expression of the housekeeping gene beta-actin, a 7-fold increased expression of C3 and a 3-fold decreased expression of HO-1 in the allograft group as compared to the control group. mRNA expression results from microdissected compartments: in the control group, C3 and HO-1 expression could only be detected in the proximal tubuli of all cases whereas all five compartments analyzed from the rejecting kidneys showed expression of the two genes. In the allografts, expression levels of the investigated genes varied considerably not only among the different compartments but between individual cases as well.


CONCLUSIONS
Laser microdissection combined with real-time RT-PCR is a feasible approach for retrospective quantitative gene expression analysis in formalin-fixed and paraffin-embedded renal allograft specimens. As shown for C3 and HO-1, cell-specific expression patterns ofpathogenetically relevant genes vary considerably between individual cases. A close correlation of morphology and cell-specific gene expression analysis will contribute to the elucidation of the complex pathogenesis of chronic renal allograft nephropathy.
Serinsöz
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Institut für Pathologie, Medizinischen Hochschule, Hannover, Germany. mengel.michael@mh-hannover.de
Bock
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Kirsch
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Haller
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Lehmann
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U
Kreipe
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H
Mengel
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D000199:Actins / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D003176:Complement C3 / Q000235:genetics / Q000378:metabolism*; D006084:Graft Rejection / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D051547:Heme Oxygenase-1; D006801:Humans; D016030:Kidney Transplantation; D053685:Laser Therapy; D008565:Membrane Proteins; D042282:Microdissection; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D003176:Complement C3; D008565:Membrane Proteins; D012333:RNA, Messenger; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1