Noninvasive diagnosis of renal-allograft rejection by measurement of messenger  RNA for perforin and granzyme B in urine.
BACKGROUND
Acute rejection is a serious and frequent complication of renal transplantation, and its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for rejection could improve the outcome of transplantation.


METHODS
We obtained 24 urine specimens from 22 renal-allograft recipients with a biopsy-confirmed episode of acute rejection and 127 samples from 63 recipients without evidence of acute rejection. RNA was isolated from the urinary cells. Messenger RNA (mRNA) encoding the cytotoxic proteins perforin and granzyme B and a constitutively expressed cyclophilin B gene were measured with the use of a competitive, quantitative polymerase chain reaction, and the level of expression was correlated with allograft status.


RESULTS
The log-transformed mean (+/-SE) levels of perforin mRNA and granzyme B mRNA, which encode cytotoxic proteins, but not the levels of constitutively expressed cyclophiiin B mRNA, were higher in the urinary cells from the 22 patients with a biopsy-confirmed episode of acute rejection than in the 63 recipients without an episode of acute rejection (perforin, 1.4+/-0.3 vs. -0.6+/-0.2 fg per microgram of total RNA; P<0.001; and granzyme B, 1.2+/-0.3 vs. -0.9+/-0.2 fg per microgram of total RNA; P<0.001). Analysis involving the receiver-operating-characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 83 percent and a specificity of 83 percent with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA, and with a sensitivity of 79 percent and a specificity of 77 percent with the use of a cutoff value of 0.4 fg of granzyme B mRNA per microgram of total RNA. Sequential urine samples were obtained from 37 patients during the first nine days after transplantation; and measurements of the levels of mRNA that encoded cytotoxic proteins identified those in whom acute rejection developed.


CONCLUSIONS
Measurement of mRNA encoding cytotoxic proteins in urinary cells offers a noninvasive means of diagnosing acute rejection of renal allografts.
Department of Medicine, Weill Medical College of Cornell University, New York, USA.
B
B
Li
C
C
Hartono
R
R
Ding
V K
VK
Sharma
R
R
Ramaswamy
B
B
Qian
D
D
Serur
J
J
Mouradian
J E
JE
Schwartz
M
M
Suthanthiran
D000208:Acute Disease; D001707:Biopsy, Needle; D021983:Cyclophilins / Q000235:genetics; D017931:DNA Primers; D005260:Female; D006084:Graft Rejection / Q000175:diagnosis* / Q000652:urine; D053804:Granzymes; D006801:Humans; D007668:Kidney / Q000473:pathology; D016030:Kidney Transplantation; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D008875:Middle Aged; D019696:Peptidylprolyl Isomerase; D054353:Perforin; D016133:Polymerase Chain Reaction; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger / Q000652:urine*; D012372:ROC Curve; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000235:genetics*
D017931:DNA Primers; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger; D054353:Perforin; C081786:cyclophilin B; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase