Expression of human heme oxygenase-1 in the thick ascending limb attenuates angiotensin II-mediated increase in oxidative injury.
BACKGROUND
Heme oxygenase-1 (HO-1) catalyzes the conversion of heme to bilirubin, carbon monoxide (CO), and free iron, thus controlling the level of cellular heme. The medullary thick ascending limb of the loop of Henle (TALH) is situated in a site of markedly diminished oxygen tension and, as such, is highly vulnerable to ischemic insult. We hypothesize that selective upregulation of HO-1 in TALH by gene transfer attenuates oxidative stress caused by angiotensin II (Ang II).


METHODS
An adenoviral vector expressing the human HO-1 under the control of the TALH-specific promoter [Na(+)-K(+)-Cl(-) cotransporter (NKCC2 promoter)] was constructed and the cell specific expression of the recombinant adenovirus was examined using several types of cells, including endothelial, vascular smooth muscle, and TALH cells. The effects of HO-1 transduction on HO-1 expression, HO activity and the response to Ang II with respect to cyclooxygenase-2 (COX-2) up-regulation and oxidative injury [growth-stimulating hormone (GSH) levels and cell death] were determined.


RESULTS
Western blot and reverse transcription-polymerase chain reaction (RT-PCR) revealed that human HO-1 was selectively expressed in primary cultured TALH cells following infection with Ad-NKCC2-HO-1. In TALH cells infected with Ad-NKCC2-HO-1, Ang II-stimulated prostaglandin E(2) (PGE(2)) levels were reduced by 40%. Ang II caused a marked decrease in GSH levels and this decrease was greatly attenuated in TALH cells transduced with Ad-NKCC2-HO-1. Moreover, Ang II-mediated DNA degradation was completely blocked by the site-specific expression of human HO-1 gene.


CONCLUSIONS
These results indicate that TALH cell survival after exposure to oxidative stress injury may be facilitated by selective upregulation of HO-1, thusly blocking inflammation and apoptosis.
Quan
Shuo
S
Department of Pharmacology, Division of Nephrology, New York Medical College, Valhalla, New York 10595, USA. nader_abraham@nymc.edu
Yang
Liming
L
Shnouda
Sylvia
S
Schwartzman
Michal L
ML
Nasjletti
Alberto
A
Goodman
Alvin I
AI
Abraham
Nader G
NG
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D004249:DNA Damage; D018076:DNA, Complementary / Q000235:genetics; D015232:Dinoprostone / Q000096:biosynthesis; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism; D006418:Heme / Q000494:pharmacology; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics* / Q000378:metabolism; D051547:Heme Oxygenase-1; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008138:Loop of Henle / Q000187:drug effects / Q000293:injuries / Q000378:metabolism*; D008565:Membrane Proteins; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics; D064466:Solute Carrier Family 12, Member 1; D014161:Transduction, Genetic
D018076:DNA, Complementary; D007527:Isoenzymes; D008565:Membrane Proteins; D011994:Recombinant Proteins; C578714:SLC12A1 protein, human; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D000804:Angiotensin II; D006418:Heme; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C105245:heme oxygenase-2; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D005978:Glutathione; D015232:Dinoprostone