Effect Of NF-κB Inhibitor PDTC To Kidney Immunogenicity In Brain Death Pigs

Kidney transplantation is a major method to treat end-stage kidney diseases. But less than 20 percent patients can get grafts because of organs shortage. Most patients have to live by hemodialysis. About half people died while waiting for grafts. Using the kidneys from brain death donors is a valuable way to solve this problem. Above 80 percent of transplanted patients from unrelated living donors can survive 3 years in the condition of compatibility of human leucocyte antigen compared with only 70 percent for the patients from brain death donors. The kidney transplantation from unrelated living donors has no superiority in surgery skills, treatments or genetics compared with which from brain death donors. So we speculate that brain death may play an important role in it. Studies had revealed that brain death, which could cause hemodynamics disorders, endocrine disequilibrium and chemical mediator release affected the function of grafts. Studies showed that major histocompatibility antigen- II in brain-dead kidneys increased. With immunogenicity heightenning, kidneys caused rejection easily. The exactly mechanism is unknown. NF-κB is a nuclear factor which mediates the expression of many cytokines and is regarded as the source of inflammatory reaction. Van Der Hoeven found the expression of NF-κB and the adhesion molecule increased in the kidneys of brain-dead rats. The infiltration of inflammatory cells leaded to renal injuries. PDTC is an antioxidant which inhibits the activation of NF-κB. Recent studies had showed its protective effects to acute severe pancreatitis and ischemic-reperfusion injuries. But it has not been reported what the mechanisms are for the increase of renal immunogenicity and whether it is related to the activation of NF-κB. It is important to to answer these questions. Though which we can improve the curative effects of kidney transplantation. This experiment will establish brain-dead model through increasing intracranial pressure in a modified, slow and intermittent way, and observe the changes of kidneys immunogenicity by using PDTC to study the effects of NF-κB on immunogenicity changes, to provide a theoretical support for raising survival rate by reducing brain-dead kidneys immunogenicity in clinic.ObjectivesTo study the effects of NF-κB on kidneys immunogenicity changes in brain-dead state through testing the variances of NF-κB and MHC- II before and after using PDTC and to provide a theoretical support for raising survival rate by reducing brain-dead kidneys immunogenicity in clinic.Materials and methods18 pigs were randomized into 3 groups: group C (control group, n=6), group B (brain death group, n=6) and group p (protective group, n=6). Brain death model was established by increasing intracranial pressure in a modified, slow and intermittent way, and the brain death state was maintained for 24h by respiration and circulation support. In group C only Foley balloon catheters were placed in intracalvarium, no brain-dead being established. In group B and group P, the serum and renal inferior part tissues were taken at 6, 12 and 24h after the brain-death models were established. The levels of TNF-α, IL-1βand IL-6 in serum were determined by ELISA method. The level of NF-κB mRNA in kidney tissues was detected by real-time fluorescence quantitative PCR. The protein levels of MHC-II and NF-κB in renal tissues were tested by immunohistochemistry. The morphologic changes were observed under light microscope. In group C, the changes of these aspects were also tested in the corresponding time points.Results1. At 6, 12 and 24h, the levels of TNF-αin group C were 11.01±0.90, 10.54±0.63, 10.95±0.76pg/ml, in group B, 20.21±1.12, 25.16±1.04, 34.99±0.88pg/ml, and in group P, 15.68±0.77,21.52±0.58, 29.44±1.51pg/ml. At 6, 12 and 24h, the levels of IL-1βin group C were 4.80±0.50, 4.89±0.49, 4.98±0.45pg/ml, in group B, 10.93±0.76, 14.80±0.68, 18.01±1.02pg/ml, and in group P, 8.00±0.64, 11.27±0.77, 14.30±0.46pg/ml. At 6, 12 and 24h, the levels of IL-6 in group C were 11.55±0.79, 11.34±0.84, 11.53±0.57pg/ml, in group B, 17.08±0.83, 25.29±1.47, 34.10±2.05pg/ml, and in group P, 14.23±0.67, 19.43±1.29, 24.77±1.52pg/ml. At 6, 12 and 24h, the changes of TNF-α, IL-1βor IL-6 in group C among the different time points were no significant difference(P>0.05). Compared with the closer together time points in group B and group P, there was statistic significance(P<0.05); compared with the three groups at 6, 12 and 24h, the group difference was significant(P<0.05).2. At 6, 12 and 24h, the transcriptional levels of NF-κB mRNA compared withβ-actin in group C are 0.0140±0.0025, 0.0139±0.0017, 0.0142±0.0019, in group B, 0.0469±0.0037, 0.0904±0.0091, 0.1533±0.0063, and in group P, 0.0246±0.0025, 0.0614±0.0068, 0.1123±0.0056. The levels in group C among the different time points were no statistic significance(P>0.05). Compared with the closed together time points in group B and group P, there was statistic significance(P<0.05); compared with the three groups at 6, 12 and 24h, the group difference was significant(P<0.05).3. The cells with positive expression of NF-κB and MHC-II proteins were low in group C and increased obviously in group B and group P. At 6, 12 and 24h, the positive cells rates of NF-κB in group C are 0.0843±0.0029, 0.0847±0.0016, 0.0877±0.0015, in group B, 0.1537±0.0015, 0.3867±0.0064, 0.5930±0.0070, and in group P, 0.1087±0.0016, 0.2807±0.0030, 0.4523±0.0010. At 6, 12 and 24h, the positive cells rates of MHC in group C are 0.0643±0.002, 0.067±0.004, 0.073±0.002, in group B, 0.122±0.003, 0.237±0.004, 0.504±0.005, and in group P, 0.099±0.0030, 0.195±0.001, 0.329±0.004. The expression levels of NF-κB or MHC- II in group C among the different time points were no significant difference(P>0.05). Compared with the closer together time points in group B and group P, there was statistic significance(P<0.05); compared with the three groups at 6, 12 and 24h, the group difference was significant(P<0.05).4. No morphological injury was ovserved under light microscope in group C. Renal tubules and renal glomerulus remained normal. In group B, no morphological change was observed at 6h, but there were slight renal proximal convoluted tubule cell edema and lumensat thinner 12h. At 24h vacuole degeneration, inflammatory leukocyte invasion and lumensat obliteration can be observed. In group P renal injury was also observed, while slighter than brain death group at the corresponding time point.Conclusions1. Brain death may cause renal morphological injury, moreover, this injury will aggravate with the brain death state was prolonged.2. Brain death may cause the increase of protein expression of MHC- II, moreover, this increase will be higher with brain death state was prolonged. Which indicates that the brain death may be the reason for renal immugenicity increasing.3. The levels of NF-κB and inflammatory factors increased after brain death. And with the time going, the levels got higher. These indicated that brain dead may result in the activation of NF-κB.4. The level of MHC- II and NF-κB in brain-dead kidneys increased consistently which indicated that the activation of NF-κB may cause the increase of renal immunogenicity.5. PDTC may decrease the expression of MHC- II in brain-dead state and lower kidney immunogenicity through inhibiting the activation of NF-κB and reducing the release of inflammatory factors. Which also proved the relation among the brain death, NF-κB and renal immugenicity.