Ischemia-Reperfusion Renal ischemia and reperfusion injury : influence of chorpromazine on renal function and lipid peroxidation

Purpose: To evaluate the influence of chlorpromazine (CPZ) on renal function and lipid peroxidation in a rat model of kidney ischemia/reperfusion injury. Methods: Forty eight Wistar rats underwent a laparotomy for hilar clamping of left kidney with a bulldog clamp for 60 minutes followed by organ reperfusion and contralateral nephrectomy. Of these, 26 received 3mg/kg of CPZ intravenously 15 minutes before renal ischemia (G-E) while the remaining 22 were used as ischemic control group (G-C). Eleven rats of G-E and 8 of G-C were followed for blood urea nitrogen and creatinine determinations before renal ischemia and at 1, 4 and 7 postoperative days. Samplings of left renal tissue were obtained at 5 minutes (5 rats from each group) and 24 hours (9 G-C and 10 of G-E) of reperfusion for malondialdehy (MDA) content determination. Controls of renal MDA content were determined in kidneys harvested from 6 additional normal rats. Results: Acute renal failure occurred in all animals but levels of BUN and creatinine were significantly lower in G-E (p<0.001). MDA content rose strikingly at 5 minutes of reperfusion in both groups (p>0.05) and returned near to normal levels 24 hours later. Conclusion: CPZ conferred partial protection of renal function to kidneys submitted to ischemia/reperfusion injury that seems to be not dependent on inhibition of lipid peroxidation.


Introduction
Ischemia/reperfusion (I/R) injury is a major cause of acute renal failure occurring after hemorrhagic shock or major cardiovascular surgery, and the most common cause of delayed graft function or organ failure. 1,2Ischemiareperfusion injury is a complex phenomenon involving not only intracellular processes but also an injurious inflammatory response that are interconnected. 34][5] ROS formed during oxidative stress can initiate lipid peroxidation, oxidize proteins to inactive states and cause DNA strand breaks, all potentially damaging to normal cellular function. 3,4Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellular membranes, and their destruction can lead to the production of toxic reactive aldehydic metabolites and cell death. 5-8A range of drugs and methods have been tested with the intention to block the pathways of cell injury at different levels and to thereby enhance viability of the kidney challenged by ischemia/ reperfusion stress with variable results, such as: heat shock (interferes with the L-arginine-nitric oxide pathway), 9 antioxidants, 10,11 dipyridamole (increases endogenous adenosine), 12,13 nicotine (inhibit cholinergic antiinflammatory pathway), 14 calcium channel blockers or antagonists, 15-18 chlorpromazine, 19,20 etc. Chorpromazine (CPZ) is a membrane stabilizer and seems to be able to preserve mitochondria integrity and to reduce phospholipids degradation of microsomal membranes of liver cell provoked by ischemia/reperfusion. 20,21 The aim of our study was to explore the effect of chlorpromazine on renal function and membrane lipid peroxidation in a rat model of renal ischemia/reperfusion.

Methods
Forty eight Wistar Rattus novergicus, weighting 200-250g, were anesthetized with an intraperitoneal injection of thiopental (20mg/kg) and underwent a laparotomy followed by dissection of the left kidney and hilar clamping with a bulldog clamp for 60 minutes followed by organ reperfusion and contralateral nephrectomy.Of these, 26 received 3mg/kg of chlorpromazine intravenously 15 minutes before renal ischemia (Group E) while the remaining 22 were used as ischemic control group (Group C).Eleven rats of Group E and 8 of Group C were followed for blood urea nitrogen (Urease-Labtest Cat 27 ® ) and creatinine (Creatinine-Labtest Cat 35 ® ) determination before renal ischemia and in the 1 st , 4 th and 7 th postoperative days.Samplings of left renal tissue were obtained at 5 minutes (5 rats from each group) and 24 hours (9 of Group C and 10 of Group E) of reperfusion for malondialdehy (MDA) content determination.Normal controls of renal MDA content were determined in left kidneys harvested from 6 additional normal rats.For tissue sampling in the described moments and at the end of experiment the animals were sacrificed by decapitation.
For the determination of MDA content kidney samples were homogenized in 9 volumes of Tris-HCl 10mM, pH 7.4, and centrifuged at 3,000 G for 10 minutes at 4ºC.The supernatant was aspirated and used to quantify MDA (expressed in µM) using a lipid peroxidation assay kit (Calbiochem, cat # 437634, Novobiochem Co, USA) and a spectrophotometer at absorbance of 586nm.
Statistical analyses were performed with the software program GraphPad Prism 4 (GraphPad Software Inc, San Diego, CA).Continuous variables among groups were compared by Kruskal-Wallis method and Dunn´s multiple comparisons test as long as their distribution did not pass the normality test or the differences among the SDs were very significant.A level of P<0.05 was set as statistical significant.

Results
The results of blood urea nitrogen (BUN) and creatinine in groups C and E at different moments of the experiment are displayed in Tables 1 and 2. In the group C renal dysfunction lasted till the 7 th postoperative day and in all moments was worse than in group E (p<0.001).In the group E, creatinine and BUN returned to normal levels on the 7 th postoperative day.
The mitochondrial MDA content found in normal kidneys and in kidneys challenged with 60 minutes of ischemia followed by 5 minutes or 24 hours of reperfusion is expressed in Table 3.The MDA rose significantly at 5 minutes of reperfusion in both groups of rats, treated or not with CPZ (p>0.05).Twenty four hours after the ischemic challenge MDA content tended to return to pre-ischemic values.

Discussion
Kidneys submitted to warm ischemia during 60 minutes exhibit functional deficit resulting in uremia as published previously by others. 22,23In the control group the BUN and creatinine levels remained high till the 7 th postoperative day even though exhibiting a tendency to return to normal values.In the group treated with CPZ the levels of BUN and creatinine were lower than in the control group in the period 1 st -4 th days, and at the 7 th day their values return to normal.Thus, CPZ in the dose of 3mg/kg conferred a partial protection of renal function to kidneys challenged with warm ischemia for 60 minutes.CPZ ability to protect tissues against ischemia/reperfusion was reported by others working with several organs such as: kidney, 22,24 liver, 19,25 nervous system, 26 heart. 27Upon ischemia/ reperfusion challenge, organ depletion of ATP causes mitochondrial dysfunction, and accumulation of intracellular sodium, calcium and ROS. 4 An associated mechanism of cell injury is the accelerated rate of phospholipid degradation in ischemic rat liver tissue, producing a loss of almost one half the total cellular phospholipid with 3 hours of ischemia, and pretreatment of the rats with CPZ (20mg/kg) completely prevented the disturbed phospholipid metabolism at the same time that it prevented the cell death associated with as much as 3 hours of ischemia. 19,28Several studies have also demonstrated that warm ischemia in kidney is associated with lipid peroxidation that leads to oxidative destruction of cellular membranes, and their destruction can lead to the production of toxic, reactive aldehydic metabolites and cell death. 1,7,8Among the aldehydic metabolites, MDA and 4-hydroxynonenal are the most important. 29,30Superficial and deep cortex immunostaining with antibody against MDA was observed after 30 minutes of warm renal ischemia and such immunostaining was observed in the absence of any histological lesions, as assessed by routine staining; after 45 and 60 minutes of warm ischemia, lipid peroxidation byproducts were detected both in the cortex and in the medulla, which is associated with 33% and 66% of rat deaths respectively. 31Thus the lipid peroxidation process is involved in kidney damage during anoxia before reperfusion, and the presence of its byproducts in renal medulla was associated with the animals' death. 31Our study confirms that lipid peroxidation occurs during anoxia since MDA content rose strikingly at 5 minutes of kidney reperfusion and tended to return to normal values 24 hours later.However, pretreatment of animals with CPZ, dose of 3mg/kg, was not able to inhibit lipid peroxidation in our model of renal ischemia which is in disagreement with the findings reported for ischemic rat liver. 19,28A possible explanation for such controversy may be based on the difference of CPZ dose employed that was of 20mg/kg in the model of liver ischemia.The protection of renal function against ischemia/reperfusion injury promoted by CPZ at a dosage of 3mg/kg seems to be not related to inhibition of lipid peroxidation.

References
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