Cold preservation of isolated rabbit proximal tubules induces radical-mediated cell injury.
BACKGROUND
Reactive oxygen species (ROS) are involved in reperfusion injury after preservation. Recent studies in isolated endothelial cells and hepatocytes suggested the occurrence of ROS-mediated injury during the period of cold incubation. In the present study, formation of ROS and subsequent cell injury were studied in freshly isolated rabbit proximal tubules (PTs).


METHODS
PTs were incubated in University of Wisconsin (UW) solution, Euro-Collins solution, or a modified Krebs-Henseleit buffer under aerobic conditions for up to 94 hr at 4 degrees C. ROS formation and cell death were assessed as lipid peroxidation (formation of thiobarbituric acid-reactive substances [TBARS]) and release of lactate dehydrogenase, respectively. The involvement of ROS was further investigated in UW solution using compounds that might interfere with ROS formation. In addition, tubules were studied under anaerobic conditions (gassing with 95% N2/5% CO2).


RESULTS
Cold preservation of rabbit PTs in any of the solutions under aerobic conditions caused progressive lipid peroxidation and concomitant cell injury. Addition to UW solution of inhibitors of ROS formation, in particular 2,2'-dipyridyl, or removal of oxygen by gassing with 95% N2/5% CO2, prevented lipid peroxidation and protected rabbit PTs against cold injury. Both the nitric oxide (NO) synthase inhibitor L-NAME and dexamethasone, which blocks the inducible NO synthase, were ineffective. The cytoprotectant glycine affected neither TBARS formation nor lactate dehydrogenase release.


CONCLUSIONS
Cold preservation of renal PTs under aerobic conditions caused cell injury even in the specially designed preservation solution UW. Cell injury is caused by iron-dependent, NO synthase-independent ROS formation.
Peters
S M
SM
Department of Internal Medicine, University Hospital Nijmegen, The Netherlands.
Rauen
U
U
Tijsen
M J
MJ
Bindels
R J
RJ
van Os
C H
CH
de Groot
H
H
Wetzels
J F
JF
D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D002021:Buffers; D016923:Cell Death / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D003080:Cold Temperature; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione; D006982:Hypertonic Solutions; D007328:Insulin; D007501:Iron; D007687:Kidney Tubules, Proximal; D008054:Lipid Peroxides / Q000378:metabolism; D019188:Organ Preservation Solutions; D011817:Rabbits; D011887:Raffinose; D017382:Reactive Oxygen Species; D014021:Tissue Preservation / Q000379:methods*
D002021:Buffers; D002614:Chelating Agents; C037096:Euro-Collins' solution; D016166:Free Radical Scavengers; D006982:Hypertonic Solutions; D007328:Insulin; D008054:Lipid Peroxides; D019188:Organ Preservation Solutions; D017382:Reactive Oxygen Species; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D007501:Iron; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose