renal ischemia - reperfusion ( ir ) injury is major cause of acute kidney injury , a common clinical problem associated with an increasing prevalence and high mortality rate ( 1 ) .
the severity of the injury depends on the duration of ischemia and subsequent reperfusion phase .
reperfusion causes damage through generation of reactive oxygen species and inflammation rather than restoration of normal function ( 2 ) .
therefore , the need for additional therapeutic modalities to prevent renal ir injury is quite urgent .
erythropoietin is a glycoprotein hormone that was originally identified as the humoral factor , which control production of erythroid precursor cells .
however , many evidence suggests that epo has several functions independent of its effects on red blood cells production .
recently , studies in vitro and vivo have shown that epo attenuates cell damage ( 3 ) .
the proper effects of the epo - related changes are not fully clearly , even though it has anti - apoptotic , antioxidative and anti - inflammatory properties .
its pro - angiogenic potential seems to be related to epo - mediated protective effect .
ischemic preconditioning ( ipc ) , defined as brief sublethal periods of ischemia followed by reperfusion before master ischemia , is a way to minimize subsequent events of ir injury ( 4 , 5 ) . researches
show that ipc regimen can protect the target organs or distant parts of organs and tissues ( 6 , 7 ) .
protective effects of ipc on ir injury have been frequently demonstrated in other organs such as skeletal muscle ( 9 ) .
antioxidants have been presented to be protective against ir interposed oxidative damage in different animal models ( 8) .
lipid peroxidation refers to the oxidative deterioration of lipids and is one of the most important sources of oxidative stress .
several experimental studies have shown that lipid peroxidation occurred in renal ir insult ( 10 ) . therefore , ros has been shown to contribute to the cellular damage induced by ir .
the aim of this study was to compare the effects of recombinant human epo and ipc on renal ischemia / reperfusion injury and their effects on the production of ros .
male wistar albino rats , weighing 220 - 270 g , were used . until the beginning of the research ,
rats were kept at room temperature ( 23c - 25c ) and 40% - 60% relative humidity on a 12/12 hour light / dark cycle and were fed with standard pellet diet and water ad libitum .
all procedures described below were performed under approval of the animal ethics committee of tehran university of medical sciences .
rats were anaesthetized by an intraperitoneal administration mixture of ketamine ( 50 mg / kg ) and xylazine ( 10 mg / kg ) .
systolic blood pressure and heart rate were recorded by a tail - cuff connected to a pulse transducer device ( mlt125/r ; ad instruments , castle hill , nsw , australia ) .
the transducer was linked to a powerlab/4sp data - acquisition system ( chart , version 5 ; ad instruments ) .
after satisfactory surgical anesthesia had been achieved , a midline laparotomy was done , in which the abdominal cavity was fully exposed .
bilateral renal pedicles were isolated carefully and clamped by non - crushing microvascular clamp to effect complete cessation of renal blood flow .
after 50 minutes , the clamps were removed to allow return of blood flow to the kidneys , and then kidneys were undergone to reperfusion for 24 hours .
the edges of the abdominal incision were joined to each other and covered by a piece of gauze soaked with warm isotonic saline ( 37c ) to prevent undue loss of body fluids .
the abdomen was irrigated with warm isotonic saline , and then the abdominal wound was closed in two layers by continuous stitches .
the ischemic preconditioning ( ipc ) was performed by three cycles of alternating 3 minutes of bilateral renal pedicles ischemia and 3 minutes reperfusion .
rats were allocated randomly into four experimental groups ( n = 6 ) : sham group , after laparotomy , rats were subjected to surgical manipulation without intervention ; ir group , rats were subjected to bilaterally renal ischemia for 50 minutes followed by 24 hours reperfusion ; ir + epo , rats were subjected to ir ( as in ir group ) , a single dose of epo ( 5000 u / kg ) was injected intraperitoneally thirty minutes before the onset of ischemia ; ir + ipc , rats were subjected to ischemic preconditioning ( ipc ) regimen before the onset of ischemia ( figure 1 ) . at the end of the surgery ,
after 24 hours reperfusion , they were anaesthetized by mixture of ketamine ( 50 mg / kg ) and xylazine ( 10 mg / kg ) and blood samples were collected from the inferior vena cava .
rats were sacrificed and their kidneys were harvested before being washed and dissected in cold normal saline .
part of the left kidney was immediately snap - frozen in liquid nitrogen and stored at 70c for renal oxidative stress status assay .
blood samples were centrifuged at 1,000 g for 15 minutes within 1 hour after collection .
plasma samples were analyzed for blood urea nitrogen ( bun ) and plasma creatinine with commercial kits by an autoanalyzer .
kidney ( 50 mg ) tissue samples were homogenized with trichloroacetic acid ( tca ) .
thiobarbituric acid ( tba ) solution was added to the supernatant and boiled for 60 minutes .
after samples were cooled down , the optical density of supernatant was measured at 532 nm . to measure sod activity , 50 mg of kidney tissue samples were homogenized in buffer phosphate .
a spectrophotometric method was used to determine renal sod activity which was based on the inhibition of a superoxide - induced nicotinamide adenine dinucleotide oxidation
kidney tissue samples ( 50 mg ) were homogenized in ethylenediaminetetraacetic acid ( edta)-buffer phosphate and tca solution .
the assay for gsh is based on the reaction of gsh with 5 , 5-dithiobis ( 2-nitrobenzoic acid ) , which produces the 2-nitro-5-thiobenzoic acid chromophore that can be monitored at 412 nm ( 13 ) .
male wistar albino rats , weighing 220 - 270 g , were used . until the beginning of the research ,
rats were kept at room temperature ( 23c - 25c ) and 40% - 60% relative humidity on a 12/12 hour light / dark cycle and were fed with standard pellet diet and water ad libitum .
all procedures described below were performed under approval of the animal ethics committee of tehran university of medical sciences .
rats were anaesthetized by an intraperitoneal administration mixture of ketamine ( 50 mg / kg ) and xylazine ( 10 mg / kg ) .
systolic blood pressure and heart rate were recorded by a tail - cuff connected to a pulse transducer device ( mlt125/r ; ad instruments , castle hill , nsw , australia ) .
the transducer was linked to a powerlab/4sp data - acquisition system ( chart , version 5 ; ad instruments ) .
after satisfactory surgical anesthesia had been achieved , a midline laparotomy was done , in which the abdominal cavity was fully exposed .
bilateral renal pedicles were isolated carefully and clamped by non - crushing microvascular clamp to effect complete cessation of renal blood flow .
after 50 minutes , the clamps were removed to allow return of blood flow to the kidneys , and then kidneys were undergone to reperfusion for 24 hours .
the edges of the abdominal incision were joined to each other and covered by a piece of gauze soaked with warm isotonic saline ( 37c ) to prevent undue loss of body fluids .
the abdomen was irrigated with warm isotonic saline , and then the abdominal wound was closed in two layers by continuous stitches .
the ischemic preconditioning ( ipc ) was performed by three cycles of alternating 3 minutes of bilateral renal pedicles ischemia and 3 minutes reperfusion .
rats were allocated randomly into four experimental groups ( n = 6 ) : sham group , after laparotomy , rats were subjected to surgical manipulation without intervention ; ir group , rats were subjected to bilaterally renal ischemia for 50 minutes followed by 24 hours reperfusion ; ir + epo , rats were subjected to ir ( as in ir group ) , a single dose of epo ( 5000 u / kg ) was injected intraperitoneally thirty minutes before the onset of ischemia ; ir + ipc , rats were subjected to ischemic preconditioning ( ipc ) regimen before the onset of ischemia ( figure 1 ) . at the end of the surgery ,
after 24 hours reperfusion , they were anaesthetized by mixture of ketamine ( 50 mg / kg ) and xylazine ( 10 mg / kg ) and blood samples were collected from the inferior vena cava .
rats were sacrificed and their kidneys were harvested before being washed and dissected in cold normal saline .
part of the left kidney was immediately snap - frozen in liquid nitrogen and stored at 70c for renal oxidative stress status assay .
blood samples were centrifuged at 1,000 g for 15 minutes within 1 hour after collection .
plasma samples were analyzed for blood urea nitrogen ( bun ) and plasma creatinine with commercial kits by an autoanalyzer .
kidney ( 50 mg ) tissue samples were homogenized with trichloroacetic acid ( tca ) .
thiobarbituric acid ( tba ) solution was added to the supernatant and boiled for 60 minutes .
after samples were cooled down , the optical density of supernatant was measured at 532 nm . to measure sod activity ,
a spectrophotometric method was used to determine renal sod activity which was based on the inhibition of a superoxide - induced nicotinamide adenine dinucleotide oxidation .
the optical density of supernatant was measured at 340 nm ( 12 ) . reduced glutathione ( gsh ) levels in kidney tissue were determined by spectrophotometery .
kidney tissue samples ( 50 mg ) were homogenized in ethylenediaminetetraacetic acid ( edta)-buffer phosphate and tca solution .
the assay for gsh is based on the reaction of gsh with 5 , 5-dithiobis ( 2-nitrobenzoic acid ) , which produces the 2-nitro-5-thiobenzoic acid chromophore that can be monitored at 412 nm ( 13 ) .
kidney ( 50 mg ) tissue samples were homogenized with trichloroacetic acid ( tca ) .
thiobarbituric acid ( tba ) solution was added to the supernatant and boiled for 60 minutes .
after samples were cooled down , the optical density of supernatant was measured at 532 nm .
to measure sod activity , 50 mg of kidney tissue samples were homogenized in buffer phosphate .
a spectrophotometric method was used to determine renal sod activity which was based on the inhibition of a superoxide - induced nicotinamide adenine dinucleotide oxidation .
kidney tissue samples ( 50 mg ) were homogenized in ethylenediaminetetraacetic acid ( edta)-buffer phosphate and tca solution .
the assay for gsh is based on the reaction of gsh with 5 , 5-dithiobis ( 2-nitrobenzoic acid ) , which produces the 2-nitro-5-thiobenzoic acid chromophore that can be monitored at 412 nm ( 13 ) .
bilateral renal ischemia ( 50 minutes ) and 24 hours reperfusion resulted in a significant increase in plasma bun compared with that in the sham group ( 22.50  3.18 vs 133.33  5.48 mg / dl respectively ; p < 0.05 ) and an increase in plasma creatinine ( 0.29  0.08 vs 2.75  0.40 mg / dl , respectively ; p < 0.05 ) .
furthermore , epo significantly decreased plasma bun and creatinine ( 64.33  8.75 mg / dl and 1.26  0.36 mg / dl ; respectively ; both p < 0.05 ) compared to that in the ir group .
ischemic preconditioning also significantly decreased plasma bun and creatinine ( 68.60  5.87 mg / dl and 1.28  0.14 mg / dl ; respectively ; both p < 0.05 ) compared to that in the ir group ( figure 2 ) .
the sham group did not undergo any further interventions ; the ischemia / reperfusion ( ir ) group was subjected to 50 minutes bilateral ischemia followed by 24 hours reperfusion .
a single dose of epo ( 5000 u / kg , i.p ) was administered 30 minutes prior to ischemia .
ischemic preconditioning consisted of three cycles of 3 minutes intermittent ir of renal pedicles applied at the beginning of the 50 minutes period of renal ischemia , followed by 24 hours reperfusion .
data are the mean  sem ( n = 6 to 9 ) . * p < 0.05 compared to the sham group ; # p < 0.05 compared to the ir group ( one - way anova followed by tukey s test ) . bilateral renal ischemia ( 50 minutes ) and 24 hours reperfusion resulted in significant increase in mda content compared to the sham group ( 1.20  0.09 vs 2.13  0.14 mol/100 mg tissue , respectively ; p < 0.05 ) .
however , epo significantly decreased mda content ( 1.56  0.1 mol/100 mg tissue ; p < 0.05 ) compared to the ir group .
ischemic preconditioning also significantly decreased mda content ( 1.54  0.07 mol/100 mg tissue p < 0.05 ) compared to the ir group ( figure 3a ) .
the sham group did not undergo any further interventions ; the ischemia / reperfusion ( ir ) group was subjected to 50 minutes bilateral ischemia followed by 24 hours reperfusion . a single dose of epo ( 5000 u / kg , i.p ) was administered 30 minutes prior to ischemia .
ischemic preconditioning consisted of three cycles of 3 minutes intermittent ir of renal pedicles applied at the beginning of the 50 minutes period of renal ischemia , followed by 24 hours reperfusion .
* p < 0.05 compared to the sham group ; # p < 0.05 compared to the ir group ( one - way anova followed by tukey s test ) .
in addition , bilateral renal ischemia ( 50 minutes ) and 24 hours reperfusion significantly decreased sod activity compared to the sham group ( 29.84  2.78 vs 9.04  1.44 u / g tissue , respectively ; p < 0.05 ) .
the sod activity in the epo group ( 22.37  2.34 u / g tissue ; p <
ischemic preconditioning also significantly increased the sod activity ( 21.48  3.22 u / g tissue ; p < 0.05 ) compared to the ir group ( figure 3b ) . compared to the sham group , bilateral renal ischemia ( 50 minutes ) and 24 hours reperfusion resulted in a significant decrease in gsh levels ( 79.63  3.5 vs 26.02  4.36 mol / g tissue , respectively ; p < 0.05 ) . the gsh level was increased in the epo group ( 67.69  4.48 mol / g tissue p < 0.05 ) compared to that in the ir group .
the gsh levels were also increased in the ipc group ( 64.74  3.42 mol / g tissue ; p < 0.05 ) compared to that in the ir group ( figure 3c ) .
abbreviations : ir , ischemia / reperfusion ; epo , erythropoietin ; ipc , ischemic preconditioning ; bun , blood urea nitrogen ; scr , serum creatinine ; mda , malondialdehyde ; sod , superoxide dismutase ; gsh , glutathione .
bilateral renal ischemia ( 50 minutes ) and 24 hours reperfusion resulted in a significant increase in plasma bun compared with that in the sham group ( 22.50  3.18 vs 133.33  5.48 mg / dl respectively ; p < 0.05 ) and an increase in plasma creatinine ( 0.29  0.08 vs 2.75  0.40 mg / dl , respectively ; p < 0.05 ) .
furthermore , epo significantly decreased plasma bun and creatinine ( 64.33  8.75 mg / dl and 1.26  0.36 mg / dl ; respectively ; both p < 0.05 ) compared to that in the ir group .
ischemic preconditioning also significantly decreased plasma bun and creatinine ( 68.60  5.87 mg / dl and 1.28  0.14 mg / dl ; respectively ; both p < 0.05 ) compared to that in the ir group ( figure 2 ) .
the sham group did not undergo any further interventions ; the ischemia / reperfusion ( ir ) group was subjected to 50 minutes bilateral ischemia followed by 24 hours reperfusion .
a single dose of epo ( 5000 u / kg , i.p ) was administered 30 minutes prior to ischemia .
ischemic preconditioning consisted of three cycles of 3 minutes intermittent ir of renal pedicles applied at the beginning of the 50 minutes period of renal ischemia , followed by 24 hours reperfusion .
data are the mean  sem ( n = 6 to 9 ) . * p < 0.05 compared to the sham group ; # p < 0.05 compared to the ir group ( one - way anova followed by tukey s test ) .
bilateral renal ischemia ( 50 minutes ) and 24 hours reperfusion resulted in significant increase in mda content compared to the sham group ( 1.20  0.09 vs 2.13  0.14 mol/100 mg tissue , respectively ; p < 0.05 ) . however , epo significantly decreased mda content ( 1.56  0.1 mol/100 mg tissue ; p < 0.05 ) compared to the ir group .
ischemic preconditioning also significantly decreased mda content ( 1.54  0.07 mol/100 mg tissue p < 0.05 ) compared to the ir group ( figure 3a ) .
the sham group did not undergo any further interventions ; the ischemia / reperfusion ( ir ) group was subjected to 50 minutes bilateral ischemia followed by 24 hours reperfusion . a single dose of epo ( 5000 u / kg , i.p ) was administered 30 minutes prior to ischemia .
ischemic preconditioning consisted of three cycles of 3 minutes intermittent ir of renal pedicles applied at the beginning of the 50 minutes period of renal ischemia , followed by 24 hours reperfusion .
data are the mean  sem ( n = 6 to 9 ) . * p < 0.05 compared to the sham group ; # p < 0.05 compared to the ir group ( one - way anova followed by tukey s test ) . in addition , bilateral renal ischemia ( 50 minutes ) and 24 hours reperfusion significantly decreased sod activity compared to the sham group ( 29.84  2.78 vs 9.04  1.44 u / g tissue , respectively ; p < 0.05 ) .
the sod activity in the epo group ( 22.37  2.34 u / g tissue ; p <
ischemic preconditioning also significantly increased the sod activity ( 21.48  3.22 u / g tissue ; p < 0.05 ) compared to the ir group ( figure 3b ) .
compared to the sham group , bilateral renal ischemia ( 50 minutes ) and 24 hours reperfusion resulted in a significant decrease in gsh levels ( 79.63  3.5 vs 26.02  4.36 mol / g tissue , respectively ; p < 0.05 ) .
the gsh level was increased in the epo group ( 67.69  4.48 mol / g tissue p < 0.05 ) compared to that in the ir group .
the gsh levels were also increased in the ipc group ( 64.74  3.42 mol / g tissue ; p < 0.05 ) compared to that in the ir group ( figure 3c ) .
abbreviations : ir , ischemia / reperfusion ; epo , erythropoietin ; ipc , ischemic preconditioning ; bun , blood urea nitrogen ; scr , serum creatinine ; mda , malondialdehyde ; sod , superoxide dismutase ; gsh , glutathione .
renal ir injury is a complicated process in which the kidney is subjected to morphological and functional damage during the ischemic phase and undergoes further insult during reperfusion . in the present study
, we used a rat model of renal ir injury ( 50 minutes ) bilaterally .
the effect of pretreatment with epo or ipc on renal function , as well as on mda ( a marker of lipid peroxidation ) and sod and gsh ( markers of antioxidants ) were investigated . in this study , we present the finding that epo pretreatment and application of ipc protect the kidney against ir injury induced by 50 minutes bilateral ischemia followed by 24 hours reperfusion . our study show that epo pretreatment and application of ipc reduce renal dysfunction ( assessed by bun and creatinine ) , oxidative stress ( assessed by sod and gsh ) , and lipid peroxidation ( assessed by mda ) .
the present study demonstrated that 50 minutes bilateral renal ir injury caused significant increases in plasma levels of bun and creatinine .
these findings confirm that ir injury of the kidney causes both glomerular and tubular dysfunctions and are in agreement with those reported by gobe et al .
the sequential events of renal ir injury include both cellular damage caused by ischemic insult and generation of reactive oxygen species which results in activated vascular endothelial cells after reperfusion ( 15 ) that cause renal cell injury . to dismiss toxic reactive oxygen species , cells have many natural defense enzyme mechanisms , including the enzymes sod and cat and the anti - oxidant molecule gsh .
an increase in free radical causes overproduction of ros during ir which may lead to the consumption and depletion of these endogenous scavenger antioxidants . in the present study ,
these observations are in accordance with previous studies that reported that renal iri is associated with decreases in sod activity ( 16 ) .
our results demonstrated that , pretreatment with epo as a single dose or application of ipc , significantly increased sod activity and gsh content . in agreement with these findings baranano and snyder ( 18 ) reported that epo increased the production of radical scavengers , and akisu et al .
( 19 ) showed that epo inhibited the iron - catalyzed reactions for generating free oxygen radicals .
the data of another study showed that epo pretreatment improved the cellular antioxidant defense system following renal ir injury ( 20 ) .
previous studies have demonstrated that , mda tissue content is also commonly used as a marker of oxidative stress in renal ir injury ( 21 ) . in the present study , we demonstrated that ir resulted in increased mda content in renal tissues and was associated with impaired kidney function .
these data are in good agreement with those of jiang et al . who found high lipid peroxidation after renal ir injury ( 22 ) .
our results demonstrated that pretreatment with epo significantly decreased the level of mda , indicating lower level of oxidative stress and subsequently less lipid peroxidation .
( 23 ) demonstrated that epo decreased the level of mda after ir injury in a rat model .
our results showed that application of ipc caused a significant reduction in mda production , indicating a reduction in lipid peroxidation and cellular damage .
( 24 ) demonstrated that , ipc significantly reversed the increase in lipid hydroperoxide levels to a considerable extent .
renal ir damage leads to extensive oxidative stress . reducing the excessive production of reactive oxygen species minimizes the secondary destruction after renal injury ( 25 ) .
erythropoietin may exert its anti - oxidative effects by stimulating endothelial nitric oxide synthase and inducing intracellular anti - oxidative mechanisms .
plentiful of reactive oxygen species during the first stage of reperfusion has been presented out as the good evidence for the pathogenesis of the tissue injury .
reactive oxygen species generation increases sharply at the onset of reperfusion , although such substances are detectable in further moments too ( 26 ) .
the ipc acts in this phase in a way that has not been completely clear , probably attenuating the oxidative stress through increased production of nitric oxide .
a limitation of the current study is the lack of long - term evaluation of the kidney function of the experimental groups .
another limitation is the lack of some groups to measure the parameters in the middle of the reperfusion period . in conclusion ,
pretreatment with epo and application of ipc significantly ameliorated the renal injury induced by bilateral renal ir .
 background : ischemia - reperfusion ( ir ) injury is one of the most common causes of renal dysfunction . 
 there is increasing evidence about the role of the reactive oxygen species ( ros ) in these injuries and endogenous antioxidants seem to have an important role in decreasing the renal tissue injury.objectives:the aim of this study was to compare the effect of recombinant human erythropoietin ( epo ) and ischemic preconditioning ( ipc ) on renal ir injury.materials and methods : twenty four male wistar rats were allocated into four experimental groups : sham - operated , ir , epo + ir , and ipc + ir . 
 rats were underwent 50 minutes bilateral ischemia followed by 24 hours reperfusion . 
 erythropoietin ( 5000 iu / kg , i.p ) was administered 30 minutes before onset of ischemia . 
 ischemic preconditioning was performed by three cycles of 3 minutes ischemia followed by 3 minutes reperfusion . 
 plasma concentrations of urea and creatinine were measured . 
 kidney samples were taken for reactive oxidative species ( ros ) measurement including superoxide dismutase ( sod ) activity , glutathione ( gsh ) contents , and malondialdehyde ( mda ) levels.results:compared to the sham group , ir led to renal dysfunction as evidenced by significantly higher plasma urea and creatinine . 
 treatment with epo or ipc decreased urea , creatinine , and renal mda levels and increased sod activity and gsh contents in the kidney.conclusions:pretreatment with epo and application of ipc significantly ameliorated the renal injury induced by bilateral renal ir . 
 however , both treatments attenuated renal dysfunction and oxidative stress in kidney tissues . 
 there were no significant differences between pretreatment with epo or application of ipc . 