Cyclooxygenase-2 expression is up-regulated in obstructed human ureter.
OBJECTIVE
Prostanoids produce significant effects on ureteral function and are synthesized by cyclooxygenase (COX) enzymes. COX is found in the 2 isoforms COX-1 (a constitutive form) and COX-2 (an inducible form). Due to the side effects associated with COX-1 inhibition there is great interest in selective COX-2 inhibition. We determined if COX-2 messenger (m)RNA and protein expression are regulated during ureteral obstruction.


METHODS
mRNA analysis was performed using excess ureteral segments from 6 patients undergoing reconstructive procedures for chronic ureteral obstruction and 8 (normal ureter) undergoing donor nephrectomy after providing informed consent. All ureteral segments were snap frozen in liquid nitrogen and stored at -70C. RNA was isolated from the segments using phenol extraction and complementary DNA was synthesized by reverse transcription with murine leukemia virus reverse transcriptase (Promega Corp., Madison, Wisconsin). Semiquantitative reverse transcriptase-polymerase chain reaction was performed using specific COX-2 gene primers with ribosomal S26 primers serving as the housekeeping gene. The polymerase chain reaction product was quantified by agarose gel electrophoresis and phospho-imaging. The ratio of COX-2-to-S26 mRNA was compared. Additional segments were homogenized and total protein was extracted, separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. These membranes were Western blotted for COX-2 and glyceraldehyde-3-phosphate dehydrogenase (housekeeping protein) with specific primary and secondary antibodies.


RESULTS
The mean ratio of COX-2-to-S26 mRNA plus or minus standard error at 20, 22 and 24 cycles of amplification was 0.22 +/- 0.04 in the 8 normal ureters compared with 1.01 +/- 0.21 in the 6 obstructed ureters (unpaired Student's t test p = 0.004). Similarly the mean ratio of COX-2-to-glyceraldehyde-3-phosphate dehydrogenase protein on immunoblotting was 0.15 +/- 0.02 in the 8 normal ureters compared with 0.59 +/- 0.10 in the 6 obstructed ureters (p = 0.003).


CONCLUSIONS
These data indicate that COX-2 mRNA and protein levels are up-regulated in chronically obstructed human ureters. Using selective COX-2 inhibitors may be useful for treating prostanoid induced effects associated with ureteral obstruction.
Nakada
Stephen Y
SY
Divisions of Urology and Transplantation, Department of Surgery, University of Wisconsin Medical School, Madison, Wisconsin, USA.
Jerde
Travis J
TJ
Jacobson
Lynn M
LM
Saban
Ricardo
R
Bjorling
Dale E
DE
Hullett
Debra A
DA
D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D051546:Cyclooxygenase 2; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D007223:Infant; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D008875:Middle Aged; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000378:metabolism; D015854:Up-Regulation; D014513:Ureter / Q000378:metabolism*; D014517:Ureteral Obstruction / Q000378:metabolism*
D007527:Isoenzymes; D008565:Membrane Proteins; D011506:Proteins; D012333:RNA, Messenger; C489203:RPS26 protein, human; D012269:Ribosomal Proteins; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases