The Role Of PI3K/AKT In Rats With Acute Kidney Injury Induced By Abdominal Sepsis

Objective:To investigate the role of PI3K/AKT in rats with acute kidney injury induced by abdominal sepsis.Methods:Adult male Sprague-Dawley(SD)rats with weight 220-250 g were selected in this study.The experimental design was separated in two parts.From one aspect,abdominal sepsis model of cecal ligation and puncture was established.A total of 70 rats were randomly divided into CLP6 h group(n=12),CLP12 h group(n=12),CLP24 h group(n=12),CLP48 h group(n=12),CLP72 h group(n=12)and sham-operated group(n=10).Sham-operated rats underwentanesthesia and laparotomy,the cecum was separated without ligation or puncture.The survival rate was recorded and the sample of abdominal aorta blood was harvested at the different point-in-time.TNF-?,IL-1?were detected by enzyme-linked immunosorbent assay(ELISA)in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group and sham-operated group.From another aspect,abdominal aorta blood and the kidney tissue were havested in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group and sham-operated group.The variation of the concentration of Cr and BUN was tested by fully automatic biochemical analyser.RT-PCR was used to determined the PI3K/AKTmRNA in kidney tissue in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group and sham-operated group.Western blot was used to verify the expression of NLRP3,ASC and caspase-1 in kidney tissue in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group and sham-operated group.Immunohistochemistry was used to verify the expression of NLRP3,ASC and caspase-1 in kidney tissue in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group and sham-operated group.IL-18 was detected by ELISA in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group and sham-operated group.Results:Part I:With the time past,the vital signs of the rats in sham-operated group were not changes obviously.And we observed that the viscera were normal when the sham-group's rats underwent laparotomy.The rats' viscera in CLP groups were adhesion and congestion prominently,especially in ileum.And the symptoms and signs were serious with time past.The inflammatory changes were remarkable,such as the increasing of abdominal effusion and cacosmia.The survival rate in each group is 100%?91.6%?83.3%?66.7%?50%?33.3%.The rats' concentration of TNF-? in serum in sham-operated group was low and stable.Compared with the sham-operated group,rats' concentration of TNF-? in serum in experimental groups was significantly increased(P<0.01)and gradually elevated with time past.The rats' concentration of IL-1?in serum in sham-operated group was low and stable.Compared with the sham-operated group,rats' concentration of IL-1? in serum in experimental groups was significantly increased(P<0.01)and gradually elevated with time past.The level of the concentration of IL-1? in serum was stable after 48 h in CLP groups.PartII:Compared with the sham-operated group,the concentration of Scr and BUN were increased significantly in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group(P<0.01)and gradually elevated with time past(Scr:45.79±7.63,66.76±6.45,68.75±7.89,79.4±5.96,80.42±8.87,82.88±9.78;BUN:7.78±1.53,12.47±1.15,14.67±0.89,18.35±2.21,20.97±2.05,24.53±1.79).After being detected by RT-PCR,we demonstrated that compared with the sham-operated group,the expression of PI3 KmRNA was significantly increased(P<0.01)and gradually elevated with time past in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group.And compared with the sham-operated group,the expression of AKTmRNA was significantly increased(P < 0.01)and gradually elevated with time past in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group.After being detected by WB,we demonstrated that compared with the sham-operated group,the expression of NLRP3?ASC?Caspase-1 was significantly increased(P<0.01)and gradually elevated with time past in CLP6 h group,CLP12 h group,CLP24 h group,CLP48 h group,CLP72 h group.And the expression of these protein was stable after 24 h in CLP groups.After being detected by IHC,the similar result was observed compared with the WB.After being detected by ELISA,we demonstrated that compared with the sham-operated group,rats' concentration of IL-18 in serum in experimental groups was significantly increased(P<0.01)and gradually elevated with time past.Then the rats' concentration of IL-18 in serum gradually declined after 24 h in experimental groups(P<0.01).Conclusion:1?The experiment confirmed that the expression of PI3K/AKT was markedly increased in rats with acute kidney injury induced by abdominal sepsis.And these data proved that the PI3K/AKT played an important role in sepsis.2?The experiment confirmed that the expression of NLRP3 inflammasome was significant increased in rats with abdominal sepsis.What is more,the expression of the downstream cytokines such as IL-1??IL-18 were significant increased.These result suggested that the NLRP3 inflammasome and IL-18 were important in acute kidney injury induced by abdominal sepsis.