| Objective: To establish a model of mice with renal ischemia reperfusion injury(IRI),the objectives of this study were to observe the changes in renal histopathology,ultrastructure,renal function,autophagic gene and autophagy-related protein expression in the presence of renal IRI in the autophagy-induced state and the non-induction state respectively,as well as the effect of hyperbaric oxygenation therapy(HBO)on its expression,discuss the effect of the occurrence and development and signaling of authophagy in renal IRI as well as its effect on renal function,and further clarify the role of autophagy in renal IRI and its impact on HBO therapy.Methods: C57BL/6 mice were randomly divided into non-autophagy induction group(non-rapamycin treatment)and autophagy induction group(rapamycin treatment).Each group was divided into normal control group,sham operation group,renal ischemia group,HBO+renal ischemia group,renal IRI group and renal IRI+HBO group,while the sham operation group,renal IRI group and renal IRI+HBO group were further divided into 1h group,3h group,6h group,12 h group and 24 h group,6 mice for each group.All mice in the autophagy-induction group were treated with rapamycin 1h before establishing renal ischemia and renal IRI model.For the normal control group,only the abdominal cavity was opened immediately before cutting both kidneys and collecting blood.For the sham operation group,the abdominal cavity was opened,bilateral renal pedicles were not blocked,both kidneys were cut and venous blood were collected at 1h,3h,6h,12 h and 24 h.For the renal ischemia group,the abdominal cavity was opened,bilateral renal pedicles were blocked for 45 min,and both kidneys were cut and venous blood was collected.For HBO+renal ischemia group,the abdominal cavity was opened after the mice were pre-treated with HBO,bilateral renal pedicles were blocked for 45 min,and bilateralkidneys were cut and venous blood was collected.For the renal IRI group,the abdominal cavity was opened,bilateral renal pedicles were blocked for 45 min and bloodstream was opened,and both kidneys were cut and venous blood was collected 1h,3h,12 h and 24 h after reperfusion respectively.For renal IRI+HBO group,the abdominal cavity was opened,bilateral renal pedicles were blocked for 45 min and bloodstream was opened,HBO treatment was given 0h,2h,5h and 23 h after reperfusion respectively,both kidneys were cut and venous blood was collected 1h after HBO treatment.HE staining was conducted to detect the expression of protein Beclin-1,LC3-II,Atg-4,PKC and PI3K-III in renal tissues,the expression of protein LC3-II was detected by Western blot,and serum creatinine(Scr)was determined by fully automatic biochemical analyzer.Results.1.Changes in renal function:(1)In non-rapamycin treatment group and rapamycin treatment group,there was no significant difference in SCr level between sham operation group and normal control group;the SCr level of renal ischemia group was obviously higher than that in normal control group and sham operation group(P<0.01);the SCr level of HBO+renal ischemia group was much lower than that of renal ischemia group(P<0.05),but obviously higher than those of normal control group and sham operation group(P<0.01);the SCr level of renal IRI group at each time point was obviously higher than those of renal ischemia group,normal control group and sham operation group(P<0.01);the SCr level of renal IRI+HBO group at each time point was remarkably higher than those of normal control group and sham-operation group,and the SCr levels of renal IRI+HBO group at 1h,3h and 6h were obviously higher than those of renal IRI group(P<0.01);there were statistically significant differences in SCr level within renal IRI group and renal IRI+HBO group at each time point(P<0.05),and SCr level increased gradually with time and achieved the maximum at 24 h.(2)Between rapamycin group and non-rapamycin group: The SCr level of rapamycin renal IRI group and renal IRI+HBO group at 1h,3h,6h,and 12 h were obviously lower than those of non-rapamycin group(P<0.05);there was no statistically significant difference in SCr level between each of the rest rapamycin group and the correspondingnon-rapamycin group.2.Changes in renal pathology:In non-rapamycin treatment group and rapamycin treatment group,the renal tissue structure was normal in both normal control group and sham operation group at each time point.In renal ischemia group,minor bleeding was noted in renal tubules,mild extravasated blood was found in glomerular capillaries,and there was mild tubular swelling.After autophagy induction,tubulointerstitial bleeding and capillary congestion of glomerular capillary were obvious,and a few renal tubular epithelial cells necrosis and debris were seen.After pretreatment with HBO,no obvious bleeding was found in glomerular and tubular mesenchyme in HBO+renal ischemia group,and the rest part showed no obvious difference compared with renal ischemia group under microscope.In renal IRI group,glomerular swelling could be noted at 1h and 3h,some glomerular lumens disappeared,a small amount of falling debris of dead epithelial cells could be noted in glomerular lumens,granular degeneration occurred in cytoplasm,glomerular mesenchyme bleeding was noted,the lesions gradually worsened with time,falling debris of some tubular epithelial cells could be noted at 12 h,large falling debris of flaky renal tubular epithelial cells could be noted at 24 h.After autophagy induction,falling debris of a large amount of renal tubular epithelial cells was found in renal tubular lumens,and protein casts increased.After pretreatment with HBO,there were a large amount of red cell casts at the same time,which was most obvious at the junction of cortex and medulla.In renal IRI+HBO group,swelling of tubular cells and interstitial hemorrhage were most obvious at1 h,the lesions at 3h and 6h were alleviated than renal IRI group,hemorrhage was relieved with time,renal tubular epithelia cell necrosis and extravasated blood in glomerular capillaries at 12 h and 24 h were alleviated than those in renal IRI group at the same time point,but tubulointerstitial hemorrhage was severer than those at the corresponding time points,hemorrhage was obvious in medulla,fibrinoid substance exudation could be seen in glomerular cyst cavity and there were a few red cell casts.3.Change of autophagosome under Transmission electron microscopy(TEM):In non-rapamycin treatment group and rapamycin treatment group,the renal tissuestructure was normal in both normal control group and sham operation group at each time point,and several autophagosomes were formed occasionally.In the renal ischemia group,some mitochondria were slightly enlarged with no well-defined membrane structure,the number of autophagosomes was slightly increased than those of normal control group and sham operation group,and the rest showed no significant difference compared with those of normal control group and sham operation group,as shown under TEM.After autophagy induction,the number of autophagosomes increased,the lesions observation showed no difference under TEM,the number of autophagosomes increased further.In renal IRI group,a large number of autophagic vacuoles and autophagosomes,autophagosomes in double-membrane structure were obvious,mitochondria and proteins encapsulated by autophagosomes were displayed clearly,the formation of lipid droplets with fatty degeneration were noted,the smooth endoplasmic reticulum was obvious,and no obvious abnormality was found in the mitochondria structure,nuclear membrane and nucleolus.In renal IRI 6h group,the number of autophagosomes was the highest,micro microvilli of renal tubular epithelial cells lost their membranous structure,but the profile was still recognizable,mitochondria were slightly enlarged,there were a few of lysosome,the number of autophagosomes was slightly decreased at 12 h and 24 h,micro microvilli on the free surface of renal tubular epithelial cells lost their membranous structure,mitochondria were slightly enlarged,some cristae fragmentation was noted,a small amount of secondary lysosomes could be found,renal tubular basilar membrane was not clear,and nuclear membrane was not clearly displayed with rugas.The above changes at 24 h were the most obvious.After autophagy induction,the number of autophagosomes increased and the lesion worsened at the corresponding time point.After treatment with HBO,the number of autophagosomes increased further,the autophagosomes was still more in 12 h and 24 h,but the lesion was alleviated and the changes were still at 24 h.4.Changes in expression of Beclin-1,LC3-II,PI3K-III,PKC and Atg-4 in renal tissues by IHC:(1)Beclin-1: Beclin-1 protein was mainly expressed in renal tubular epithelial cell cytoplasm and tubular interstitium,and a few was expressed in the glomeruli.The relative quantification result revealed that:(1)In non-rapamycin treatment group and rapamycin treatment group,there was no significant difference in the expression of Beclin-1 protein between normal control group,sham operation group and renal ischemia group;The expression of Beclin-1 protein was distinctly decreased in the HBO+renal ischemia group compare with the the normal control group,sham operation group and renal ischemia group(P<0.01).The expression of Beclin-1 protein increased distinctly in renal IRI group of non-rapamycin treatment group at 6 and 12 h compare with the normal control group,sham operation group and renal ischemia group(P<0.01),while the expression of Beclin-1 protein increased distinctly in renal IRI group of rapamycin treatment group at 1h,3h,and 6h compare with the normal control group,sham operation group and renal ischemia group(P<0.01).In non-rapamycin treatment group and rapamycin treatment group,the expression of Beclin-1 protein increased distinctly in renal IRI+HBO group at 1h and 3h compare with the normal control group,and sham operation group(P<0.01),The expression of Beclin-1 protein decreased distinctly in renal IRI+HBO group at 12 h and 24 h compare with the normal control group,and sham operation group(P<0.01).The expression of Beclin-1 protein decreased distinctly in renal IRI+HBO group of rapamycin treatment group at 1and 24 h compare with the normal control group and sham operation group(P<0.01),and increased distinctly in renal IRI+HBO group at 6h compare with the normal control group and sham operation group(P<0.01).The expression of Beclin-1 protein decreased distinctly in renal IRI+HBO group at 1h,3h and 6h compare with the renal IRI group(P<0.01).In non-rapamycin treatment group and rapamycin treatment group,the expression of Beclin-1 protein was no significant difference in the sham operation group at each time point(P>0.05);in both renal IRI group and renal IRI+HBO group,there was statistical difference in the expression of Beclin-1 protein at 3h,6h,12 h and 24h(P<0.01),and the expression of Beclin-1 protein increased gradually with the prolongation of time,reached the highest at 6 h,and then gradually decreased with time.(2)Between non-rapamycin treatment group and rapamycin pretreatment group: There was no statistical difference in the expression of Beclin-1 protein between normal control group of rapamycin pretreatment group and sham operation group of non-rapamycin treatment group,the expression of Beclin-1 protein increased distinctly in renal ischemia group and HBO+renal ischemia group of rapamycin pretreatment group compare with therenal ischemia group and HBO+renal ischemia group of the non-rapamycin treatment group(P<0.01).The expression of Beclin-1 protein increased distinctly in both renal IRI group and renal IRI+HBO group of rapamycin pretreatment group at 1h,3h and 6h,and decreased distinctly at 12 h and 24 h compare with the renal IRI group and renal IRI+HBO group of non-rapamycin treatment group(P<0.01).(2)LC3-II: LC3-II protein was mainly in the cytoplasm of renal tubular epithelial cell and very slightly in the cytoplasm of glomerular epithelial cell.Relative quantification results:(1)In non-rapamycin treatment group and rapamycin treatment group,there was no significant difference in the expression of LC3-II protein between normal control group and sham operation group.In the non-rapamycin treatment group,there was no significant difference in the expression of LC3-II protein between normal control group,sham operation group and renal ischemia group.In the rapamycin treatment group,the expression of LC3-II protein was distinctly increased in the renal ischemia group compare with the the normal control group,and sham operation group(P<0.01).In non-rapamycin treatment group and rapamycin treatment group,the expression of LC3-II protein was distinctly increased in the HBO+renal ischemia group compare with the the normal control group,sham operation group and renal ischemia group(P<0.01);the expression of LC3-II protein was distinctly increased in the renal IRI group compare with the the normal control group,sham operation group and renal ischemia group(P<0.01);the expression of LC3-II protein was distinctly increased in the HBO+renal IRI group compare with the the normal control group,sham operation group and renal IRI group(P<0.01);in the sham operation group,there was no significant difference in the expression of LC3-II protein at each time point;the expression of LC3-II in renal IRI group and renal IRI+HBO group decreased gradually with time,reached the lowest in 3h,and gradually increased with time,reached the highest level in 24h(P<0.01).(2)Between non-rapamycin treatment group and rapamycin pretreatment group: The expression of LC3-II protein increased distinctly in each group and corresponding time group of rapamycin pretreatment group than that in non-rapamycin treatment group(P<0.01).(3)PI3K-III: PI3K-III protein was expressed mainly in the cytoplasm of renal tubularepithelial cell.Relative quantification results:(1)In non-rapamycin treatment group and rapamycin treatment group,there was no significant difference in the expression of PI3K-III protein between normal control group and sham operation group;the expression of PI3K-III protein was distinctly incerased in the renal ischemia group compare with the normal control group and sham operation group(P<0.01).In the non-rapamycin treatment group,there was no significant difference in the expression of PI3K-III protein in the HBO+renal ischemia group compare with the the normal control group,sham operation group.In the rapamycin treatment group,the expression of PI3K-III protein was distinctly incerased in both renal ischemia group and HBO+renal ischemia group compare with the normal control group and sham operation group(P<0.01).In the non-rapamycin treatment group,the expression of PI3K-III protein was distinctly decreased in the HBO+renal ischemia group compare with the renal ischemia group(P<0.01).In the non-rapamycin treatment group,there was no significant difference in the expression of PI3K-III protein between renal ischemia group and HBO+renal ischemia group.In non-rapamycin treatment group and rapamycin treatment group,the expression of PI3K-III protein increased distinctly in renal IRI group at 6h and12 h compare with the normal control group and sham operation group(P<0.01),the expression of PI3K-III protein decreased distinctly in renal IRI group at 1h and 24 h,and increased distinctly at 6h compare with the renal ischemia group(P<0.01).In the rapamycin treatment group,the expression of PI3K-III protein increased distinctly in renal IRI group at 1h,3h and 6h,and increased distinctly at 12 h and 24 h compare with the renal ischemia group(P<0.01).In non-rapamycin treatment group and rapamycin treatment group,the expression of PI3K-III protein increased distinctly in the HBO+renal IRI group at 3h,6h and 12 h compare with the normal control group and sham operation group(P<0.01),there was no significant difference in the expression of PI3K-III protein between the renal IRI group and the HBO+renal IRI group.In the rapamycin treatment group,the expression of PI3K-III protein increased distinctly in renal IRI+HBO group compare with the normal control group,sham operation group and renal IRI group(P<0.01),In non-rapamycin treatment group and rapamycin treatment group,in the sham operationgroup,there was no significant difference in the expression of PI3K-III protein at each time point;In both renal IRI group and renal IRI+HBO group,there was statistical difference in the expression of PI3K-III protein(P<0.01).(2)Between non-rapamycin treatment group and rapamycin pretreatment group: The expression of PI3K-III protein decreased distinctly in normal control group,sham operation group and renal ischemia group of rapamycin pretreatment group than that in the non-rapamycin treatment group(P<0.01),the expression of PI3K-III protein increased distinctly in renal IRI+HBO group of rapamycin pretreatment rapamycin pretreatment group than that in the non-rapamycin treatment group(P<0.01),the expression of PI3K-III protein increased distinctly in renal IRI group of rapamycin pretreatment group at 1h,12 h and 24 h,and decreased distinctly at 3h and 6h compare with the renal IRI group of non-rapamycin treatment group(P<0.05),the expression of PI3K-III protein increased distinctly in renal IRI+HBO group of rapamycin pretreatment at 1h,3h,12 h and 24 h,and decreased distinctly at 6h compare with the renal IRI+HBO group of non-rapamycin treatment group(P<0.05).(4)PKC: PKC protein was expressed mainly in the glomerular and renal tubulointerstitial.Relative quantification results:(1)In the non-rapamycin treatment group,there was no significant difference in the expression of PKC protein among the normal control group,sham operation group,renal ischemia group and HBO+renal ischemia group.In the rapamycin pretreatment group,there was no significant difference in the expression of PKC protein between normal control group and sham operation group,the expression of PKC protein was distinctly incerased in the renal ischemia group compare with the normal control group and sham operation group(P<0.01),there was no significant difference in the expression of PKC protein between normal control group,sham operation group and HBO+renal ischemia group;the expression of PKC protein decreased distinctly in the HBO+renal ischemia group compare with the renal ischemia group(P<0.01).In non-rapamycin treatment group and rapamycin treatment group,the expression of PKC protein increased distinctly in renal IRI group at 3h,6h and 12 h,and decreased distinctly at 24 h compare with the normalcontrol group and sham operation group(P<0.01);the expression of PKC protein decreased distinctly in renal IRI+HBO group at 3h,6h and 12 h,and increased distinctly at24 h compare with the renal IRI group(P<0.01);there was no significant difference in the expression of PKC protein at each time point;In both renal IRI group and renal IRI+HBO group,there was statistical difference in the expression of PKC protein(P<0.01),and increased gradually with time,reached the highest in 6h,and gradually decreased with time,reached the lowest level in 24 h.(2)Between non-rapamycin treatment group and rapamycin pretreatment group: There was no significant difference in the expression of PKC protein in normal control group,sham operation group,and renal IRI+HBO group of rapamycin pretreatment group than that in the non-rapamycin treatment group,the expression of PKC protein increased distinctly in both renal ischemia group and renal IRI group of rapamycin pretreatment group at 1h,3h,6h and 12 h than that non-rapamycin treatment group(P<0.05),the expression of PKC protein decreased distinctly in the renal IRI+HBO group of rapamycin pretreatment group at 3h,and decreased distinctly at 24 h than that in non-rapamycin treatment group(P<0.01).(5)Atg-4: Atg-4 protein was expressed mainly in the cytoplasm of renal tubular epithelial cell.Relative quantification results:(1)In the rapamycin pretreatment group,there was no significant difference in the expression of Atg-4 protein between normal control group and sham operation group;the expression of Atg-4 protein increased distinctly in renal ischemia group and HBO+renal ischemia group compare with the normal control group and sham operation group(P<0.01);the expression of Atg-4 protein decreased distinctly in HBO+renal ischemia group compare with the renal ischemia group(P<0.01).In the rapamycin pretreatment group,there was no significant difference in the expression of Atg-4 protein among the normal control group,sham operation group and HBO+renal ischemia group.The expression of Atg-4 protein was distinctly decreased in the renal ischemia group compare with the normal control group and sham operation group(P<0.01).The expression of Atg-4 protein increased distinctly in the HBO+renal ischemia group compare with the renal ischemia group(P<0.01).In the rapamycin pretreatment group,the expression of Atg-4 proteinincreased distinctly in renal IRI group compare with the normal control group and sham operation group(P<0.01),and decreased distinctly compare with the renal ischemia group(P<0.01).In the non-rapamycin treatment group,the expression of Atg-4 protein increased distinctly in renal IRI group at 1h and 3h,and decreased distinctly at 6h and 24 h compare with the normal control group and sham operation group(P<0.01).In the rapamycin pretreatment group,the expression of Atg-4 protein increased distinctly in renal IRI+HBO group compare with the normal control group and sham operation group(P<0.01),and increased distinctly in renal IRI+HBO group at 1h,6h and 12 h,and decreased distinctly at3 h and 24 h compare with the renal IRI group(P<0.01).In the rapamycin treatment group,the expression of Atg-4 protein decreased distinctly in renal IRI+HBO group at 1h,3h and6 h,and increased distinctly at 24 h compare with the normal control group and sham operation group(P<0.01),and the expression of Atg-4 protein decreased distinctly in renal IRI+HBO group at 1h and 6h compare with the renal IRI group(P<0.01).In non-rapamycin treatment group and rapamycin treatment group,there was no significant difference in the expression of Atg-4 protein at each time point.In the non-rapamycin treatment group,there was statistical difference in the expression of Atg-4 protein at each time point of renal IRI group(P<0.01),and the expression of Atg-4 protein increased gradually with time,reached the highest in 3h,and gradually decreased with time.In the rapamycin treatment group,the expression of Atg-4 protein of 1h in renal IRI group increased distinctly than of 3h,6h,12 h and 24h(P<0.01),and decreased gradually with time,reached the lowest in 6h,and the expression of Atg-4 protein of 12 h and 24 h in renal IRI group increased distinctly than of 6h(P<0.01).In non-rapamycin treatment group and rapamycin treatment group,there was statistical difference in the expression of Atg-4protein at each time point of renal IRI+HBO group(P<0.01).(2)Between non-rapamycin treatment group and rapamycin pretreatment group: The expression of Atg-4 protein increased distinctly in both normal control group and sham operation group of rapamycin pretreatment group than that in non-rapamycin treatment group(P<0.01),the expression of Atg-4 protein decreased distinctly in other group of rapamycin pretreatment group than that in non-rapamycin treatment group(P<0.01).5.Changes in expression of LC3-II in renal tissues by Western blot:(1)In non-rapamycin treatment group and rapamycin treatment group,there was no significant difference in the expression of LC3-II protein between sham operation groupand normal control group.In the non-rapamycin treatment group,the expression of LC3-II protein in renal ischemia group and HBO+renal ischemia group was obviously higher than sham operation group and normal control group(P<0.05);the expression of LC3-II protein in HBO+renal ischemia group was obviously higher than renal ischemia group(P<0.05).In the rapamycin treatment group,the expression of LC3-II protein in renal ischemia group and HBO+renal ischemia group was obviously lower than sham operation group and normal control group(P<0.01),and there was no significant difference in the expression of LC3-II protein between renal ischemia group and HBO+renal ischemia group.In the non-rapamycin treatment group,the expression of LC3-II protein in renal IRI group at each time point was obviously lower than sham operation group and normal control group(P<0.01),and in renal IRI group at 3h was obviously lower renal ischemia group(P<0.01).In the rapamycin treatment group,the expression of LC3-II protein in renal IRI group at each time point was obviously lower than sham operation group,normal control group and renal ischemia group(P<0.05).In non-rapamycin treatment group and rapamycin treatment group,the expression of LC3-II protein in renal IRI+HBO group at each time point was obviously higher than sham operation group,normal control group and renal IRI group(P<0.05).The expression of LC3-II protein in sham operation group at each time point showed not significant difference.The expression of LC3-II protein in renal IRI group and renal IRI+HBO group at each time point showed statistically significant differences(P<0.01),and decreased gradually with time and reached the minimum at 3h,and then gradually increased with time,reaching the maximum at 24 h.(2)Between non-rapamycin treatment group and rapamycin pretreatment group:protein The expression of LC3-II protein in each other rapamycin pretreatment group was remarkably higher than that of the corresponding non-rapamycin treatment group(P<0.05).6.Correlation analysis between SCr and LC3-II The correlation analysis showed that the correlation coefficient of mouse SCr and renal tissue protein LC3-II expression quantity r was 0.7156 in non-rapamycin group,and0.6719 in rapamycin group,both showing a positive correlation(all P<0.01).Conclusions:1.HBO pretreatment can protect against renal ischemic injury.2.Early HBO therapy can alleviate renal IRI,and the protective effect of HBO reduces with time.3.The activation of autophagy aggravated renal IRI,and HBO therapy protects renal IRI while activating autophagy.That is to say,HBO therapy played double-edged sword role in renal IRI.4.In renal IRI,HBO therapy activates autophagy mainly through Beclin1-Atg14-Vps34-Vps15 signaling pathways.5.In renal IRI,HBO therapy plays an increasingly obviously role in inducing autophagy with time.This is an important reason why the protective role of HBO therapy attenuates in the later period of renal IRI.This also shows how HBO therapy has a double-edged effect in renal IRI. |
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