| With the rapid development of organ transplant technology, renal transplantation(RTx) has now become an effective therapeutic modality of choice for ESRD(Endstage renal disease, ESRD) patients. Ischemia/reperfusion (I/R) is an unavoidableconsequence of the renal transplant procedure. Histologic signs of IR injury in earlyrenal allograft biopsies are associated with a proinflammatory state and have beenlinked to decreased early and late allograft function. NF-kB is regarded as one of themost important and best-characterized transcription factors. Upon stimulation, theinhibitor of NF-kB becomes degraded, and NF-kB releases and translocates to thenucleus where NF-kB binds to its consensus sequence in target genes. NF-kBup-regulates the expression of many genes, most of which encode proteins that playcrucial and often determining roles in the processes of inflammation.Sung et alreported that NF-kB activation occurred during ischemia and reached its peak after15min of reperfusion, suggesting that NF-kB plays a major role in the initiation ofinflammationRecent studies have found that Erythropoietin (EPO) has its erythropoieticeffects, and also exhibits powerful tissue-protective effects against IRI in a widerange of organs including kidney, heart liver, and central nervous system. In renalI/R models it has been shown that administration of EPO before reperfusion as wellas after reperfusion can be cytoprotective. Protective EPO treatment has pronouncedanti-inflammatory and antiapoptotic capacities.These effects are dose dependent.EPO can activate multiple intracellular signaling, including mitogenactivated proteinkinase (MAPK), c-Jun N-terminal Kinase (JNK), and phosphatidylinositol3-kinasesignaling cascades, and induces the subsequent transcription of NF-kB.The proposedcytoprotective mechanism is binding of EPO to a tissue protective receptor (TPR).The heteromeric EPOR2-βcR2receptor complex consisting of two EPOreceptors and two beta common receptors (EPOR2-βcR2). The binding affinity of theclassic, erythropoietic complex of two EPO receptors for EPO is distinctly larger thanthe binding affinity of the protective EPOR2-βcR2complex. Thus，to induce localtissue protection, considerably higher systemic doses of EPO are required than thenormal therapeutic doses used for stimulation of erythropoiesis genes. But High doseEpo administration also induces serious side effects such as the risk of cardiovascularevents rate increased, an increased risk of thrombosis, the growth of tumor cells,which limits its wide application in clinical on protection organization. M Brines et alshowed that protective receptor subunit β cR in mouse cardiomyocytes (CD131),EpoR and βcR comprise a tissue-protective heteroreceptor to protect tissue，but nothematopoietic signaling pathway, To prevent these cardiovascular adverse effects,EPO derivatives have been developed which only activate the protectiveEPOR2-βcR2complex and do not stimulate erythropoiesis. ARA290is the newestgeneration EPO analogue. It is a small synthetic peptide, which selectively binds tothe EPOR2-βcR2complex. It has already been shown that ARA290, also known aspyroglutamate helices B-surface peptide (pHBSP), is not erythropoietic.The tissueprotective effects of ARA290have demonstrated using a number of animal models,including models of renal ischemia-reperfusion injury, and brain ischemia-reperfusion.Although the research of ARA290has achieved initial progress, and showspowerful tissue-protective effects, but the application of ARA290pretreatment ofdonor kidney and observe the effect of the initial graft injury has not been reportedafter transplantation. The aim of this study was to explore administration of ARA290in UW organ preservation solution, under4℃, and the establishment of allogenickidney transplantation in rats after transplantation, the renal protective effect,expression of nephritic factor mRNA transplantation by molecular biology methodfor the detection of transplantation and the possible mechanism of NF-κ B signal, toexplore the use of erythropoietin treatment of renal transplantation donor derivedpeptide ARA290, improve the quality of donor, reduce transplantation of early renalinjury, obtained better therapeutic effect of renal transplantation. Part1In vitro research Role of erythropoietin analogueARA290in HUVEC induced by H/R injury and the NF-κBmechanism researchObjectiveTo investigate the protection of ARA290,a non-erythropoietic EPO derivative, on thebinding activity of NF-κB and the expression of adhesion molecular and chemokinesof HUVEC induced by H/R (hypoxia/reoxygenation) injury.Methods1. The HUVEC H/R injury model was built. The HUVEC monolayers wererandomly divided into4groups：Control,H/R,ARA290,UW,all groups,except controlgroup, were subjected to hypoxia for6h in UW at4℃followed by4hreoxygenation,respectively.2. The survival rate of cells was detected by MTT chromatometry and theapoptosis was analyzed with flow cytometry.3. The DNA binding abitility of NF-κB was measured by EMSA.The geneexpression of ICAM-1, VCAM-1, P-selectin, IL-6, MCP-1were detected by RT-PCR.Result1. The cell survival rate of Control group was97.3±6.8%, and of H/R group,which decreased significantly, was only71.4±5.9%,；there was significant differencebetween the UW group and control group (P<0.01); The cell survival rate of ARA290group was slightly lower than that of control group, the survival rate was89.5±7.1%，(P<0.05), there was statistically significant difference compared with controlgroup, but was significantly higher than that of UW group and H/R group (P<0.01),there was statistically significant differences. The cell survival rate of UW Group was76.3±6%， but there was not significant difference between the UW group and H/Rgroup (P>0.05). 2. The apoptosis rate of H/R，control group，ARA290group and UW group wasrespectively13.13±0.94%,1.15±0.18%,5.34±0.61%and11.45±0.71%. Therewere statistically significant difference between ARA290pretreatment group andthe other three groups (P<0.05).3. RT-PCR results showed that,the mRNA expression of ICAM-1, VCAM-1,IL-6, MCP-1and P-selectin in ARA290group compared with the H/R groupdecreased significantly, decreased by (65.19±3.18)%,(71.53±5.13)%,(68.53±4.81)%,(51.09±4.17)%,(45.24±3.79)%, the difference was significant (P<0.01)；and the mRNA expression of UW group was expressed in the endothelial cells wasnot inhibited, slightly lower than group H/R，there was no statistical differencebetween two groups (P>0.05);The expression of mRNA in H/R group compared withControl group, were significantly upregulated (10.58±1.17) times,(22.03±3.74)%,(13.29±1.87)%,(29.13±3.15)%,(19.15±2.18) times, with statistically significantdifferences (P<0.01).4.. EMSA showed that RDU Control group was1.20±0.11; however, it was58.31±5.17in H/R group, there was significant difference compared with controlgroup and ARA290group (P<0.01). RDU of ARA290group was8.69±1.14,significantly lower than that in UW group，which was50.61±3.73, there wassignificant difference statistically (P<0.01). At the same time in the specificcompetition experiments that in the H/R group, adding unlabelled probe andunlabeled nonspecific probe SP-1competition, the RDU were1.55±0.23and54.3±1.60.Conclusion1. The preservation of HUVEC under hypothermic and hypoxia conditionfollowed by reoxygenation upregulates the gene expression of adhesion moleculesand chemokines.2. Erythropoietin analogue ARA290can show protective effect in HUVEC induced byH/R injury under hypothermic condition. And it can inhibit the apoptosis,downregulates cell adhesion molecule and chemokine mRNA expression. 3. Erythropoietin analogue ARA290preconditioning can inhibit NF-κ B DNAbinding activity Part2In vivo research Erythropoietin analogue ARA290attenuates the initiating injury of renal allografts in rat andthe Mechaminsm researchObjectiveTo explore the use of Erythropoietin analogue ARA290preconditioning renaldonor and improve the quality of donor, attenuates the initiating injury of renalallografts in rat.Methods1. To establish the rat kidney transplantation model. The rats were randomlydivided into3groups. Sham group: the SD rats underwent the sham operation and thekidney was harvested after24h. UW roup: The donor kidney was stored for6h underof hypothermic and hypoxia condition in UW, then the donor kidney was transplantedinto a Wistar recipient.The allograft was harvested at24h postransplantation.ARA290group: The donor kidney was stored for6h in UW containing10nmol/KgARA290under hypothermic and hypoxia condition, then the donor kidney wastransplanted into a Wistar recipient. The allograft was harvested at24hpostransplantation.2. To observe the protective effect of ARA290on the morphology of renalallografts in rat;3.To observe the effects of ARA290on the inflammatory cells invasion renalallografts by Immunohistochemical method.4. The gene expression of ICAM-1VCAM-1MCP-1RANTES in renal allografts were detected by RT-PCR；The count and distribution of ED1-postive cellsin renal allograft were evaluated by immunohistological study.5.The effect of ARA290on the activity of NF-κB in renal allografts weremeasured by EMSA；Renal allograft structure was assessed by light microscopy.Result1. Serum creatinine values for the ARA290group was180.28±32.06μ mmol/L,while the UW group was319.48±35.02μ mmol/L (P<0.01), it was32.15±7.08μmmol/L in Sham group, while it in ARA group was significantly lower than that inUW group (P<0.01); similarly, blood urea nitrogen values ARA290group weresignificantly lower than the UW group (19.48±3.81vs31.19±9.17mmol/L, P<0.01),and the values in UW group were significantly higher than the Sham group (31.19±9.17vs.4.21±0.69mmol/L, P <0.01)2. Histopathological changes in renal allografts were assessed in a maskedprotocol. The tissue damage is serious in UW group. A prominent feature wast thatthe main manifestation of renal tubular structure destroyed, epithelial cell swelling,desquamation, brush border loss, nuclear pyknosis, tubular dilation, occlusion, andinflammatory cells infiltration,.These changes were attenuated by treatment withARA290.3. Numerous macrophages were detected in the tubulointerstitium in UW group.Treatment of rats with ARA290was associated with a significant reduction ininterstitial macrophage infiltration.4. The gene expression of ICAM-1, VCAM-1, MCP-1and RANTES was greatlyincreased in UW group (p>0.05); it was significantly inhibited byARA290(p<0.05)compared to UW group.5.The RDU was0.54±0.30in Sham group; and it was51.35±3.18in UW group;Compared to UW group, the RDU was1.16±0.28(p<0.01)in ARA290.Conclusion1.The Erythropoietin analogue ARA290could efficiently protect the kidney from initiating injury. It significantly ameliorated the renal dysfunction andreduced a significant reduction in interstitial macrophage infiltration.And the graftmorphology can be well pretected.also.2.The gene expression of ICAM-1, VCAM-1, MCP-1and RANTES wassignificantly inhibited by ARA2903.The reno-protective by ARA290was related to attenuation of NF-kB. |
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