Nitric oxide induces apoptosis in renal tubular epithelial cells through activation of caspase-8.
The susceptibility or resistance of tubular epithelial cells (TEC) to apoptosis is pivotal to the long-term maintenance of kidney function following episodes of inflammation, such as graft rejection. TEC apoptosis can occur with ischemia as well as with proinflammatory cytokines and nitric oxide (NO), produced by infiltrating mononuclear cells. TEC can also produce abundant amounts of NO during inflammation but the role and regulation of NO-induced injury of TEC are not well understood. Apoptosis in TEC in vitro was determined by FACS analysis with annexin-V and propidium iodide staining. NO in culture supernatants was measured by Greiss reagent, and protein expression of inducible NO synthetase (NOS2/iNOS) and caspase-8 was examined by Western blot analysis. Here, we showed that murine TEC produced abundant amounts of NO in response to proinflammatory cytokines (IFN-gamma/TNF-alpha) through upregulation of NOS2, and inhibition of endogenous NO production by l-NMMA reduced TEC apoptosis in cytokine-stimulated cultures. Addition of exogenous NO (sodium nitroprusside) induced TEC apoptosis as well as caspase-8 activation in a dose-dependent manner. The key role of caspase-8 in NO-induced TEC apoptosis was demonstrated by that NO-induced TEC apoptosis can be blocked by caspase-8 inhibition using z-IETD-fmk, caspase-8 silencing with shRNA or by overexpressing the endogenous caspase-8 inhibitor c-FLIP (cellular Flice-inhibitory protein). In conclusion, endogenous NO from NOS2 activity as well as exogenous NO can contribute to renal injury through apoptosis of TEC. Activation of caspase-8 plays a central role in NO-induced apoptosis and caspase-8 inhibition may be an important therapeutic target during renal inflammation.
Du
Caigan
C
Division of Nephrology, Dept. of Medicine, The Univ. of Western Ontario, Univ.-Campus, 339 Windermere Road, London, Ontario, Canada. dcaigan@uwo.ca
Guan
Qiunong
Q
Diao
Hong
H
Yin
Ziqin
Z
Jevnikar
Anthony M
AM
D000818:Animals; D017209:Apoptosis; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000502:physiology; D006084:Graft Rejection; D007249:Inflammation; D007672:Kidney Cortex / Q000502:physiology; D016030:Kidney Transplantation; D007684:Kidney Tubules / Q000166:cytology / Q000502:physiology*; D051379:Mice; D009569:Nitric Oxide / Q000494:pharmacology*
D009569:Nitric Oxide; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases