| In recent years,with the continuous improvement of new immunosuppressive agentsand clinical immunosuppressive scheme, the incidence rate of acute rejection after renaltransplantation has decreased significantly, but the long-term survival rate hasn’tsignificantly improved. The main reason for the late loss of graft function is chronicallograft nephropathy (CAN), accounting for about40%of renal function loss.Thedevelopment of chronic allograft nephropathy, progressive and irreversible, will eventuallydevelop into graft function loss(chronic renal failure, CRF)(end stage renal disease,ESRD). Being unavoidable in the process of renal transplantation, ischemia reperfusioninjury stimulates the inflammation, resulting in chronic allograft nephropathy. Chronicallograft nephropathy mainly leads to for renal tubular epithelial cell atrophy, renalinterstitial fibrosis.Its pathological changes and inflammatory stimuli are inseparable.The first chapter Serum Hepcidin expression and its clinical significance in patientsafter renal transplantationObjective: To observe the clinical significance of serum Hepcidin in kidneytransplantation patients after ischemia reperfusion, to observe the role of Hepcidin in acuteischemia reperfusion injury after renal transplantation.Methods: We collected the peripheral blood of the patients at different time points beforeand after renal transplantation. ELISA was used to detect plasma IL-6, Hepcidin, sTfR(transferrin receptor), Cr (serum creatinine) level, then we analysed the correlation ofHepcidin and acute inflammation and reperfusion injury.Results: After allograft renal transplantation, the serum Hepcidin increased with statistically significant difference; and the serum Hepcidin had correlation with theexpression of IL-6, sTfR, Hb, there is significant difference; with the recovery of the renalfunction after renal transplantation, serum Hepcidin decreased, the ratio of serumcreatinine (Cr) and serum Hepcidin decreased as well, there was statistically significantdifference.Conclusion: Ischemia reperfusion injury can affect the expression of Hepcidin. Theexpression of Hepcidin positively correlates with the inflammatory state and the degree ofrenal function injury.The second chapter Hepcidin expression in rat renal ischemia reperfusion injuryand its significanceObjective: To observe the effect of renal ischemia reperfusion injury (IRI) on rat liver andHepcidin, IL-6, serum creatinine (Cr) and other indexes, and analyze its clinicalsignificance.Methods:40adult male SD rats were randomly divided into ischemia reperfusion group(IR) and control group (Control). According to the different time of reperfusion, ischemiareperfusion group were divided into3groups, respectively6h,24h,48h,10rats in eachgroup, and control group had10rats. Ischemia reperfusion group had resection of the rightkidney, blocked the left renal artery blood flow completely, and recovered it after45min.Liver, kidney specimens, venous blood were taken in6h,24h,48h respectively afteroperation. After45min of left and right kidney exposure, the control group closed theabdominal cavity. Liver, kidney specimens, venous blood were taken in6h,24h,48hrespectively after operation. In each group, Hepcidin levels in the liver of rats weredetected by Semi quantitative RT-PCR method, enzyme linked immunosorbent assay(ELISA) was adopted to detect the expression of hepcidin, IL-6in serum of rats, and serumcreatinine (Cr) was detected in all specimens. Pathological examination was performed onthe kidney of rats, and statistical analysis was adopted to research its correlation andclinical significance. Results: Hepcidin expression level in the liver and serum of renal ischemia reperfusiongroup was significantly higher than the control group (P<0.05),24hours to reach thehighest, then with the prolongation of reperfusion time expression content decreased; IL-6of renal ischemia reperfusion group was higher than control group (P<0.05). In reperfusiongroup, Hepcidin expression positively correlated with serum IL-6; reperfusion group,serum creatinine (Cr) expression was higher than that in control group (P<0.05), and alsopositively correlated with Hepcidin, renal Pathological examination found inflammatorycells reached the highest in24hours.Conclusion: Renal ischemia reperfusion injury can affect the expression of Hepcidin,which positively correlates with the inflammatory state and the degree of renal functioninjury. Therefore, Hepcidin can be used as a marker to reflect the renal ischemiareperfusion injury degree, and has clinical research value in changes of iron metabolisminduced by the renal ischemia reperfusion injury.The third part Hepcidin expression in chronic allograft nephropathy of rats andits significanceObjective: To investigate the Hepcidin expression changes in chronic allograftnephropathy rats (CAN) animal models and clinical significance.Methods: F344inbred rats as donors, inbred Lewis rats as receptor, renal transplantation,20CAN rats were established in the model; before the establishment of model, venousblood and urine were collected respectively1month,2months and4months afteroperation to test renal function. Enzyme linked immunosorbent assay (ELISA) wasused to detect serum, urine hepcidin, serum IL-6, EPO expression, and10rats werekilled respectively in2months and4months after transplantation, then pathologicalchanges of transplanted renal tissue in rats of each group were observed. Statisticalanalysis was adopted to explore its clinical significance and correlation.Results: the serum Hepcidin, IL-6expression gradually increased with the grafting time,urinary hepcidin excretion decreased gradually, EPO expression gradually reduced. The indicators before and after surgery had statistically significant difference (P<0.05). Aftertransplantation, in rats serum creatinine (Cr), blood urea nitrogen (BUN) increasedgradually, and serum creatinine (Cr) correlation with serum Hepcidin was positive. Aftertransplantation, serum Hepcidin had positive correlation with IL-6expression, hadnegative correlation with EPO; renal pathology, HE staining were in accordance withCAN pathologic change.Conclusion:1. With impaired renal allograft function, the micro inflammation in ratsincreases. The changes of Hepcidin expression in rat models of CAN correlate with renalfunction.2. With the changes of time and the inflammatory state, Hepcidin can be usedas markers to reflect renal injury.The fourth chapter The molecular mechanisms of IL-6regulating hepcidin in renaltubular epithelial (HK-2) cellsObjective: Through the stimulation of IL-6on HK-2cells, to observe the effect on thetransdifferentiation of HK-2cells and the regulation of expression of Hepcidin in HK-2cells, and to discuss the molecular mechanism of IL-6in HK-2cells regulating Hepcidin.Methods: HK-2cells were isolated, cultured, and divided into2groups,(1)control group;(2) medium added with IL-6, with final concentration of10,25,50ng/ml concentration ofIL-6added into HK-2cells, we observed the changes of HK-2cell morphology after48H,immunohistochemistry was used to detect the expression of E-cadherin, aSMA expression.ELISA method was used to determine the expression of Hepcidin of culture supernatant;Westblot method was used to determine the intracellular STAT3expression, and weanalysed the correlation of IL-6on Hepcidin, STAT3expression and HK-2celltransdifferentiation.Results: After48hours of different concentration of IL-6acting on HK-2cells, E-cadherindecreased, aSMA increased, precipitated the transdifferentiation of HK-2cells, the culturesupernatant Hepcidin concentration significantly increased, the expression of p-STAT3increased; with the correlation between the expression of IL-6on Hepcidin and p-STAT3. Conclusion：(1) IL-6can stimulate HK-2cell fibroblasts to differentiate into myocytes.(2)IL-6in HK-2cells may be through the activation of JAK/STAT signal pathway inducedupregulation of p-STAT3, increased expression of Hepcidin.(3)The Hepcidin expressionchanges correlate with HK-2Cells Transdifferentiation ability, indicating that hepcidinplays an important role in renal interstitial fibrosis, affects chronic allograft nephropathy,and can be used as markers of renal function damage. |
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