| Objective: Pre-irrigation kidney with saline is used to remove circular “cell population” and other injurious substances from the ischemic renal blood vessels.The aim of this study is to explore effects of pre-irrigation on renal ischemia-reperfusion injury in rats,and investigate the expression of macrophage isoforms(M1 and M2)in ischemic kidney after removing “inflammation cells”.Methods:18 healthy SD male rats are divided into sham operation group(Sham),ischemia-reperfusion injury group(IR),pre-irrigation+ischemia-reperfusion injury group(PI+IR)(6 rats per group).Sham Group: After the laparotomy,separating the left and right kidney pedicles carefully,and then remove the right kidney.IR Group: the left kidney pedicle is clipped for ischemia for 60 minutes and then reperfused for 24 hours,then the right kidney is removed.PI+IR Group: pre-irrigation the left kidney to clear the renal blood vessels absolutely,the rest actions are the same as IR Group.24 hours after surgery,the serum and left kidney specimens are collected to detect the levels of serum creatinine(Scr),urea(BUN),renal pathology and infiltrated inflammatory cells by HE staining.Determinating the levels of renal antioxidant catalase(CAT)and superoxide dismutase(SOD)by kit method.Detecting the expression of apoptosis-proliferating proteins macrophages and their chemoattractant-relatedproteins(HIF-α,Caspase3,PCNA,CD68,iNOS,IL-10,TNF-α,MIF,MCP-1)by Immunohistochemistry(IHC-P).Detection the expressions of MIF and MCP-1 mRNA by RT-PCR.Detect the expression of Caspase3,PCNA,TNF-α,MCP-1,CD68,iNOS and CD206 by Western blotting(WB).Results:(1)18 rats were used to suffer the surgery,16 rats survived successfully,but 2 rats died.The 2 death models were made again,and the death rate was 10%.(2)Pathological results: There was no obvious pathological damage in Sham Group,but IR Group suffered severe damages,a large number of inflammatory cells infiltrated in the tissue,and the tubular structure was destroyed and disordered with interstitial edema.The tubular epithelial cells were swollen,detached or even necrotic.In the PI+ IR Group,just a handful of inflammatory cells infiltration were observed.Only a small amount of renal tubular epithelial cells and renal interstitial were destroyed.Pathological scores: The pathological score of IR Group was significantly higher than that of Sham Group(P<0.05).Compared with IR Group,the pathological score of PI+IR Group was decreased(P<0.05).(3)Kidney function: The levels of Scr and BUN were lower in Sham Group and significantly higher in IR Group(P<0.05).The levels of Scr and BUN in PI+IR Group were lower than that of IR Group(P<0.05).(4)The levels of serum and tissue CAT were low in Sham Group,but tissue SOD level was high in it.Compared with Sham Group,the levels of serum and tissue CAT were higher in IR Group,but tissue SOD level was lower(P<0.05).Compared with IR Group,CAT and SOD levels were higher in PI+IR Group significantly(P<0.05).(5)The expressions of HIF-1α,Caspase3 and PCNA in Sham Group were lower,but were significantly higher in IR Group(P<0.05).Compared with IR Group,the expressions of HIF-1α and Caspase3 in PI+IR Group were lower(P<0.05),the expression of PCNA in PI+IR Group was significantly up-regulated(P<0.05).(6)The expressions of MCP-1,MIF and their mRNA,and the expression of TNF-α in Sham Group were decreased,up-regulated in IR Group significantly(P<0.05).Compared with IR Group,all results of protein expression in PI+IR Group were decreased(P <0.05).(7)The number of CD68+ macrophages and its protein expression in Sham Group were decreased,but up-regulated higher in IR Group(P<0.05).Compared with IR Group,the results above was lower in PI+IR Group(P< 0.05).(8)The expressions of iNOS,CD206 and IL-10 were decreased in Sham Group but up-regulated significantly in IR Group(P<0.05).Compared with IR Group,the expression of iNOS in PI +IR Group was decreased(P<0.05),the expressions of CD206 and IL-10 in PI+IR Group were significantly increased(P<0.05).Conclusion:(1)Pre-irrigation can improve the pathological and functional damage of the RIRI model.(2)Pre-irrigation could alleviate antioxidant function,accelerate the proliferation of renal tubular epithelial cells,improve hypoxic microenvironment and reduce inflammation of the RIRI model.(3)Pre-irrigation can reduce the intrarenal infiltration of macrophages and the expression of pro-inflammatory M1,while promote the expression of anti-inflammatory M2. |
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