Perforin, Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection: analyses on serial biopsies suggest methodological issues.
BACKGROUND
The Perforin-Granzyme B and Fas/Fas Ligand apoptotic mechanisms are involved in the development of acute renal rejection (AR). We describe our experience of analyzing the expression of cytotoxic T-lymphotoxins (CTL) in biopsies and peripheral blood leukocytes (PBL) for the diagnosis of AR.


METHODS
We analyzed Perforin (P), Granzyme B (GB) and Fas Ligand (FL) expression in 68 renal biopsies and 64 PBL using comparative kinetic RT-PCR and, for GAPDH and FL, we also replicated with real-time RT-PCR. The levels of expression were measured in different groups, such as T0 (biopsies before reperfusion and PBL in recipient before the transplant [Tx]), Td (biopsies and PBL collected for clinical purposes) and Tp (biopsies and PBL two months after Tx).


RESULTS
A higher CTL expression was seen in non-rejecting (NR) biopsies in the first 2 months after Tx. P and FL were significantly more expressed during AR in all biopsies and in Td, while P remained upregulated in Tp. In PBL, there was no significant increase in CTL transcription during AR. A variable expression of CTL emerged in all T0 biopsies.


CONCLUSIONS
Two lytic pathways are activated in biopsies when AR occurs shortly after Tx, whereas the P/GB mechanism prevails if it occurs later on. Only P and FL in biopsies might be able to predict AR diagnosis, but with a considerable variability in each sample, possibly due to the small portion of tissue core, which may be inadequate for molecular diagnosis. CTL expression in PBL does not correlate with histological AR.
Graziotto
Romina
R
Clinic of Nephrology, Department of Medical and Surgical Science, University of Padova, Padova, Italy. romina.graziotto@unipd.it
Del Prete
Dorella
D
Rigotti
Paolo
P
Anglani
Franca
F
Baldan
Nicola
N
Furian
Lucrezia
L
Valente
Marialuisa
M
Antonello
Augusto
A
Marchini
Francesco
F
D'Angelo
Angela
A
Gambaro
Giovanni
G
D000208:Acute Disease; D000328:Adult; D000368:Aged; D001706:Biopsy; D053222:Fas Ligand Protein; D005260:Female; D006084:Graft Rejection / Q000175:diagnosis*; D053804:Granzymes; D006801:Humans; D007668:Kidney / Q000473:pathology; D016030:Kidney Transplantation / Q000276:immunology*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D008875:Middle Aged; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000235:genetics*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014184:Transplantation, Homologous; D048069:Tumor Necrosis Factors / Q000235:genetics*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D048069:Tumor Necrosis Factors; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases