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Authors Nakada SY, Jerde TJ, Jacobson LM, Saban R, Bjorling DE, Hullett DA
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Journal J. Urol. Volume: 168 Issue: 3 Pages: 1226-9
Publish Date 2002 Sep
PubMed ID 12187272

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.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.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).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. Copyright © 2017 The Board of Regents of the University of Wisconsin System