Immuno-protection And Their Mechanisms Of Prostaglandin E1 And Low Molecular Weight Heparin Against The Experimental Renal Ischemia Reperfusion Injury In Rat

OBJECTIVE:To approach the immuno-protection and their possible mechanisms of prostaglandin E1(PGE1) and low molecular weight heparins(LMWH) against the experimental renal ischemia reperfusion injury(IRI) in rat model.IRI model was establis hed and treated with PGE1 and LMWH respectively.The expression of proinflammatory cytokines(TNF-aαIFN-γ) and anti-inflammatory cytokines(IL-4,IL-10) as well as polymorphonuclear neutrophils-derived intercellular adhesion molecular-1(ICAM-1), kidney injury molecule 1(KIM-1),angiotensinⅡ(ANGⅡ),angiotensinⅡreceptorⅠ(ATⅠR),thrombomodulin(TM),monocyte chemoattractant protein-1(MCP-1) and nuclear factor-κB(NF-κB) were estimated either in protein or mRNA level.METHODS:In part 1,45 healthy 8-weeks-old Wistar rats were divided into 3 groups according to defferent intervention methods(Sham-operated control,IRI control and PGE1 treated).Meanwhile,3 groups were stratified further according to the postoperative observation periods(1 h,3 h,6 h and 24 h).The right kidney was removed, the renal artery of left kidney in IRI control group and PGE1 treated group were occluded with a vascular clamp for 60 minutes.PGE1 treatedgroup received a PGE1 injection at the dose of 25 ng/kg after the left renal artery was occluded for 5 min.Other groups were treated with equal doses of normal saline.Blood and tissue specimen were collected at 1,3, 6 and 24 h respectively.Levels of serum creatinine(Scr) and blood urea nitrogen(BUN) were analysed.TNF-α,IFN-γ,,IL-4 and IL-10 were detected by ELISA in each group. Expressions in mRNA level of TNF-α,IL-10,MCP-1,NF-κB,KIM-1,ANGⅡ,ATⅠR, ICAM-1 were measured by real time PCR(RT-PCR).Flow cytometry was used to examine the expression of ICAM-1.Immunohistochemisal method was also applied for observation the expression of MCP-1,NF-κB and TM in renal tissue specimen.In part 2,65 healthy 8-weeks-old Wistar rats were also divided randomly into following groups:Sham-operated control group,IRI control group and LMWH treatment group.Meanwhile,3 groups were stratified further according to the postoperative observation periods(1 h,3 h,6 h,24 h,48 h and 72 h).Right kidney removed,the renal artery of left kidney in IRI control group and LMWH treatment group were occluded with a vascular clamp for 60 minutes.Instead of PGE1,we injected LMWH(500 U/kg) in rats' inferior vena at 30 minutes before reperfusion.Other groups treated with equal doses of normal saline.Blood and tissue specimen were collected at 1,3,6,24,48 and 72 h respectively.Levels of serum Scr and blood BUN were analysed.TNF-α,IFN-γ,,IL-4 and IL-10 were detected by ELISA in each group.Expressions in mRNA level of TNF-α, IL-10,MCP-1,NF-κB,KIM-1,ANGⅡ,ATⅠR,ICAM-1 were measured by RT-PCR. Flow cytometry was used to examine the expression of ICAM-1.Immuno histochemisal method was also applied for obvervation of the expression of MCP-1,NF-κB and TM in renal tissue specimen.RESULTS:1.PGE1 treatment made TNF-αat the point of 3 h and 6 h,IL-10, MCP-1 at 1 h and 3 h,statistical significant increased compared to AKI group.While there were no statistically significance at 3 h and 6 h within AKI group.2.Compared to AKI group,PGE1 treated group shows much slight appearances in renal tubular epithelial cell intumesce and protein cast formation.Glomerulum ectasia and hyperemia,renal tubular epithelial intumesces,matrix angiectasia and cellular cast could be seen in the IRI 24 h group.Immunohistochemistry shows that the level of MCP-1 and AngⅡwere declined obviously in PGE1 treated group(P＜0.01).3.24 h after reperfusion,the levels of serum Cr and BUN in IRI group and LMWH treatment group were higher than the Sham operated group(P＜0.01).Yet there was no significance in IRI group and LMWH treatment group(P＞0.05).4.After LMWH treatment,The expression of ICAM-1 in PMNs reduced 72.2%,48.3 %and 61.4%at IR 6,24 and 48 h(P＜0.01)5.24 h after reperfusion,serum TNF-αsecreted by mononuclear macrophages and IFN-γby Th1 cell in LMWH treatment group were lower than in IRI group(P＜0.01), Meanwhile,the levels of serum IL-4 and IL-10 secreted by Th2 cell in LMWH treatment group were higher than IRI group(P＜0.01).6.The renal damage of LMWH treated group was much less marked compared to that of IRI group as observed under the light microscope.7.Compared to IRI group,The expression of MCP-1 was significantly decreased at IR 6 h,24 h in LMWH treated group(P＜0.01).LMWH treatment reduced the expression of NF-κB significantly in glomcrulus of IRI group from 24.45%to 12.23%at IR 6 h after injury(P＜0.01).Meanwhile,the level of NF-κB in renal tubule decreased from 45.20%to 20.00%.While the expression of ATⅠR were increased. CONCLUSIONS:1.We establis hed the rat ischemia acute renal failure model successfully by the right kidney removement and the left renal artery occluded with a vascular clamp for 60 minutes.IRI may elevate the expression of proinflammatory cytokines in rat plasma,activates ICAM-1 to promote immunoreaction between leucocyte and renal tubular epithelial cell,and lead to AKI.2.PGE1 exhibits the inhibition to the secretion of the proinflammatory cytokines TNF-α,IFN-γ,and elevates the level of anti-inflammatory cytokines IL-4 and IL-10, leading to the depression of the over-inmmuoreaction.PGE1 could prevent platelets from releasing TM,and it was benefit to reduce platelet aggregation and to protect endothelial cell function.3.LMWH also inhibits the secretion of the proinflammatory cytokines TNF-α,IFN-γand elevates the level of anti-inflammatory cytokines IL-4 and IL-10.It also down-regulates the expression of NF-κB,MCP-1 and ICAM-1 which are benefit for rebuilding the steady state of immune responses and to protect the renal function.