Abstract
Oxidative stress has been implicated to play a major role in multiorgan dysfunction during sepsis. To study the mechanism of oxidant generation in acute kidney injury (AKI) during sepsis, we developed an in vitro model of sepsis using primary cultures of mouse cortical tubular epithelial cells exposed to serum (2.5–10%) collected from mice at 4 h after induction of sepsis by cecal ligation and puncture (CLP) or Sham (no sepsis). CLP serum produced a concentration-dependent increase in nitric oxide (NO) (nitrate + nitrite) release at 6 h and cytotoxicity (lactate dehydrogenase release) at 18 h compared with Sham serum treatment. Before cytotoxicity there was a decrease in mitochondrial membrane potential, which was followed by increased superoxide and peroxynitrite levels compared with Sham serum. The role of oxidants was evaluated by using the superoxide dismutase mimetic and peroxynitrite scavenger manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin tetratosylate hydroxide (MnTmPyP). MnTmPyP (10–100 μM) produced a concentration-dependent preservation of ATP and protection against cytotoxicity. MnTmPyP blocked mitochondrial superoxide and peroxynitrite generation produced by CLP serum but had no effect on NO levels. Although MnTmPyP did not block the initial CLP serum-induced fall in mitochondrial membrane potential, it allowed mitochondrial membrane potential to recover. Data from this in vitro model suggest a time-dependent generation of mitochondrial oxidants, mitochondrial dysfunction, and renal tubular epithelial cell injury and support the therapeutic potential of manganese porphyrin compounds in preventing sepsis-induced AKI.
Footnotes
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK075991] and the University of Arkansas for Medical Sciences Translational Research Institute, which is supported by the National Institutes of Health National Center for Research Resources [Grant UL1-RR029884].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- AKI
- acute kidney injury
- CLP
- cecal ligation and puncture
- RNS
- reactive nitrogen species
- ROS
- reactive oxygen species
- MnTmPyP
- Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin tetratosylate hydroxide
- DHR
- 2-(3,6-diamino-9H-xanthen-9-yl)-benzoic acid methyl ester
- PmCTE
- primary murine cortical tubular epithelial cells
- l-NMMA
- l-NG-monomethyl arginine citrate
- 1400W
- N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride
- DPI
- diphenyleneiodonium chloride
- JC-1
- 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide
- DAR-4M AM
- 3′,6′-Bis(dimethylamino)-9-[2-acetomethoxycarbonyl-3-amino-4-(N-methylamino)phenyl]xanthylium iodide
- LDH
- lactate dehydrogenase
- NO
- nitric oxide
- NOS
- NO synthase
- iNOS
- inducible NOS
- ONOO−
- oxidant peroxynitrite
- ANOVA
- analysis of variance
- DMEM
- Dulbecco's modified Eagle's medium
- PBS
- phosphate-buffered saline
- SOD
- superoxide dismutase.
- Received May 9, 2011.
- Accepted October 18, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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