| Objective Chronic allograft nephropathy (CAN) is the greatest obstacle to achieve long-term allograft survival. In recent years, data from clinical and experiment studies have shown that different nonimmune mechanisms are the most common factors (about 50-60%) attributed to CAN, whereas chronic rejection results only about 20% in CAN. The rest 20%-30% of CAN are caused by nephrotoxicity of calcineurin inhibitors (CNIs). Among the nonimmune mechanisms, ischemia reperfusion injury (IRI) may play an important role in development of CAN. It has a significant importance and good potency of clinical use to look for a safe and effective drug from the traditional Chinese medicine in combination with other immunosuppressants for prevention and treatment of CAN and to investigate the mechanisms of IRI causing CAN. Based on our previous studies, the present study foci on investigation of mechanism of IRI causing CAN and the protective effects of the Yisheng injection.Methods (1) The closed colony strain rats SD as well as Wistar were used as donor and recipient, respectively. Orthotopic kidney transplantation wasperformed following the procedure of Kamada with our modification. Before the transplantation, the kidney was preserved for one hour in 0~4°C sodium chloride solution for reinforcement of the injury of cold ischemia. In order to prevent acute rejection, CsA was used by pouring down stomach with the dosage of 10mg/(kg.d) for 10 days. The serum creatinine level and pathological changes of transplant kidney according to the Banff Standards were observed at the 2nd, 4th, 6th, 8th and 12th weeks post-transplantation. (2) Human umbilical endothelial cell line (ECV-304) was cultured first in RPMI medium 1640 without glucose under 100% N2 for 0.5 hours, then in RPMI medium 1640 with glucose under normal conditions for 6 or 12 hours to mimic IRI of ECs for post-transplantation. 0.5mg/ml Yisheng injection and Tiopronin injection were added into the medium, respectively. The immunohistochemistry was used to comprehend localization and expression of Smad2, Smad7 and TGF-& in the endothelial cell in different study groups. The special primers and TaqMan probes of Smads and TGF-/3i gene were synthesized to compare the expression of the corresponding genes in all groups. (3) The closed colony strain rats SD as well as Wistar were used as donor and recipient, respectively. Orthotopic kidney transplantation was performed following the established rat model of transplant kidney-sclerosis accelerated by prolonged cold ischemia time. Depended on the different given medicine, all of experimental rats were divided into four groups. Group A only received CsA 10mg/(kg.d) for lOd. For other groups^ C\ D), besides of CsA(10mg/(kg.d))X10d , rats were received either different dosage of Yisheng injection (4mg/(kg.d) or 8mg/(kg.d)) or MMF(20mg/kg.d) till different study ending, respectively. Group E was pseudo-operation controlgroup(only ligated the autologous right renal blood vessels). The serum creatinine level and pathological changes of transplant kidney were observed at the 4th, 8th and 12th weeks post-transplantation. The imrnunohistochemistry and real-time quantitative PCR were used to detect localization and expression of Smad2, Smad7 and TGF-/3i in transplant kidney. Results (1) A new SD-*Wistar rat model of transplant kidney-sclerosis accelerated by prolonged cold ischemia time was successfully built. After transplantation, the serum creatinine level of recipient rats in Group A was increased obviously since the 6th week and became uremia at the 12th week. There were no signs of acute rejection both clinically and pathologically for all the study groups. The typical pathologic changes of CAN, including vascular and glomerular sclerosis as well as interstitial fibrosis, were markedly appeared at the 8th week. Comparing to the control group, which was not treated with reinforcement of ischemic injury, the differences of SCr and pathology in IRI reinforcemental group at all the time points after 6th week were statistically significant. (2)Human endothelial cell line (ECs)' anoxia-reoxygenation injury (ART) model was built in vitro. After treated with ARI, the morphological changes of endothelial cell were observed, the ECs turning round, contracting and exfoliating. The expression of Smad2 and TGF-/3) was significantly increased in endothelial cells (ECs), while the expression of Smad7 significantly decreased. The positive expressions of target genes were mainly located in the cytoplasm of ECs. Yisheng injection and Tiopronin injection could prevent these changes and keep ECs with nearly normal morphology and function. After treated with Yisheng or Tiopronin injection, the expressions of TGF-/3] and Smad2 were significantly decreasedin ECs and up-regulated expression of Smad7 was observed. (3) SD-— Wistar orthotopic rat allograft kidney-sclerosis model was successfully built. The up-regulated expression of TGF-/3i and Smad2 and the down-regulated expression of Smad7 in group A were statistically significant when compared with other groups. The expression of TGF-/3i and Smad2 was strongly positive in transplant kidney tubular epithelial cell, interstitial cell and glomeruli whereas the expression of Smad7 was weak. Thickening of endovascular could be markly inhibited by high dosage of Yisheng injection and by MMF. Compared with the high dosage Yisheng injection group, the elevation of SCr and severity of the pathological changes in low dosage group at all time points after the 8th week were statistically significant. It showed that the protective effects of the Yisheng injection had a dose-dependent fashion. Conclusions (1) It is simple and feasible to establish a CAN rat model using SD-? Wistar, which can be accelerated through reinforcement of the cold ischemic injury. (2) IRI can damage the graft vessels and parenchyma, especially EC, in conjunction with the up-regulated expression of TGF-/3] and Smad2 and the down-regulated expression of Smad7, then initiates alone or synergizes with immune factors to accelerate the occurrence and progression of CAN. (3)Yisheng injection may play a protective role on IRI to protect ECs of graft vessels. It can decrease the expression of TGF-/3i and Smad2 and block the down-regulated expression of Smad7, which can postpone and lessen CAN. Yisheng injection is likely to play an important role by synergizing with other immunosuppressants for treatment of CAN in future.
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