Early upregulation of iNOS mRNA expression and increase in NO metabolites in pressurized renal epithelial cells.
Pressure is an important physiological regulator, but under abnormal conditions it may be a critical factor in the onset and progression of disease in many organs. In vivo, proximal tubular epithelial cells are subjected to pressure as a result of ureteral obstruction, which may influence the production of nitric oxide (NO), a ubiquitous multifunctional cytokine. To directly explore the effect of pressure on the expression and activity of NO synthase (NOS) in cultured proximal tubular epithelial cells, a novel pressure apparatus was developed. Cells were subjected to pressures of 20-120 mmHg over time (5 min-72 h). RT-PCR demonstrated an increase in inducible NOS (iNOS) and sGC, while endothelial NOS remained unchanged. Real-time PCR (qPCR) confirmed an earlier induction of iNOS transcript subjected to 60 mmHg compared with cytokine mix. iNOS protein expression was significantly increased following 60 mmHg of pressure for 24 h. Use of nuclear factor-kappaB inhibitors was shown to prevent the increase in iNOS expression following 60 mmHg for 2 h. NO and cGMP were increased with the application of pressure. The addition of the irreversible iNOS inhibitor (1400W) was shown to prevent this increase. We demonstrate that with the use of a simply designed apparatus, pressure led to an extremely early induction of iNOS and a rapid activation of NOS activity to increase NO and cGMP in proximal tubule epithelial cells. The rapid effects of pressure on iNOS may have important implications in the obstructed kidney.
Broadbelt
Nalini V
NV
Dept. of Urology, Institute for Pediatric Urology, Weill Cornell Medical Center, New York, NY 10021, USA.
Stahl
Peter J
PJ
Chen
Jie
J
Mizrahi
Moshe
M
Lal
Amit
A
Bozkurt
Alper
A
Poppas
Dix P
DP
Felsen
Diane
D
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D006152:Cyclic GMP / Q000096:biosynthesis; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D011312:Pressure; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D016328:NF-kappa B; D012333:RNA, Messenger; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D006152:Cyclic GMP