The Significance Of Endothelial Microparticles(EMP) In The Differential Diagnosis Of Acute Rejection And Of Chronic Cyclosporine Nephropathy After Rat Renal Transplantation

Objective: To study the of endothelial microparticles (EMP) in the differential diagnosis of acute rejection (AR) and chronic cyclosporine nephropathy(CCN) after rat renal transplantation and to explore the possible role of EMP in the pathogenesis of chronic cyclosporine nephropathy, the function of EMP on monocyte-macrophage cells was observed in vitro.Methods: Part I: 1.Establish the model of rat renal transplantation: In reference to the existing methods of rat renal transplantation models have been constructed, and our laboratory conditions, we improved the method to suture renal artery and venous and we established the model of rat orthotopic kidney transplantation successful. 2. Animal grouping and specimen collection: (1) Control-7days(n=9): Donor and recipient are the SD rats. (2)AR 7days group(n=8): Donor are Wistar rats and recipient are the SD rats. (3)CsA -7days group (n=9): Donor and recipient are the SD rats. (4)CsA -28days group(CCN, n=8): Donor and recipient are the SD rats. At the end of experimental time, 5ml anticoagulant blood was taken through the aorta and plasma with EMP was obtained by high-speed centrifugation, preserved at―80℃, at the same time, the donor kidney were removed and fixed in 10% neutral formaldehyde. 3. The pathological changes of the donor kidney was detected by HE staining, according to the Banff 97 standard, to determine that whether the established models is success or not. 4. Phenotypic analysis of EMP in plasma by FCM: to detect the differences of percentage of the CD144~+CD62E~+ EMP and the CD144+ perforin+ EMP in the total APC-CD144-positive EMP. The result is expressed by CD144+CD62E+ EMP% and CD144+perforin+ EMP%. Part II: 1. Identification the presence of infiltration of monocytemacrophages in CCN as described in the literature: To detected ED-1 antigen expression differences by immunohistochemistry the CsA-28days(CCN models) and the control group (Control-7days) in animal models established in Part I. 2. EMP generation and counting in vitro: EMP were got by cultured human umbilical vein endothelial cells(EAhy926) stimulated by CsA, and the number of CD144 positive EMP was calculated by flow cytometry. 3. The effect of the EMP on the human monocytic cell line (THP-1 cells): The experiment was grouped by the ratio between the number of THP-1 cells and EMP, 50μg/ml LPS as a positive control group and D-Hanks as a negative control group, the group are:①Control group (adding an equal volume of D-Hanks )②low concentrations of EMP group(1:25 EMP group)③Moderate concentration of EMP group (1:50 EMP group);④High concentrations of EMP group (1:100 EMP group);⑤50μg/L LPS positive control group. The level of TNF-αsecreted by THP-1 cells was detected by ELISA and the levels of MCP-1mRNA, HLA-DR mRNA, CCR2 mRNA expressed by THP-1 cells was detected by RT-PCR. The Statistics differences of the results among the groups were anlyzed by SPSS 18.0, P<0.05.Result: Part I: The models of rat orthotopic renal transplantation was successfully established, the pathological structure shows that the AR model and the CCN model meet the diagnostic criteria of the literature. 2. The result of CD144+CD62E+EMP%(χ±SD): (1)Control-7days group(n=9) (28.11±6.37)%; (2)AR 7days group(n=8): (60.75±5.90)%; (3)CsA -7days group(n=9): (33.78±7.82)%; (4)CsA -28days group(n=8): (75.37±8.00)%, The basic trend is (1)>(2)>(3)≥(1). 3. The result of CD144+perforin+EMP% (χ±SD): (1)Control-7days group(n=9):(10.67±2.24)%; (2)AR 7days group(n=8): (20.37±2.77)%; (3) CsA -7days group(n=9): (11.44±3.17)%; (4)CsA-28days group(n=8): (15.38±3.02)%. The basic trend is: (2)>(4)≥(3)≥(1)。Part II: 1. Compared with the Control group, a large number of ED-1 positive monocyte-macrophage cells infiltration were found in renal biopsy of the rat model of cyclosporine nephropathy(CCN group), and the result is consistent with the results described in the literature. 2. The results of the level of TNF-α, MCP-1mRNA, HLA-DR mRNA, CCR2 mRNA expressed by THP-1 cells: Compared with the Control group, their levels were all improved with the increased number of EMP, and the differences of 1:50 EMP group and 1:100 EMP group were significant, P<0.05,but the difference of the 1:25 EMP group was not significant.Conclusion:1. The study in vivo showed that the expression of the CD144+CD62E+EMP and the CD144+perforin+EMP in plasma between acute rejection and chronic cyclosporine nephropathy after rat renal transplant is significant different. Compared with CCN models, after renal transplantation, the percentage of the CD144+CD62E+EMP in the total APC-CD144-positive EMP is lower (P<0.05) and the percentage of the CD144~+perforin~+ EMP is higher (P<0.05) in AR models. The phenotypic differences are valuable in the differential diagnosis of acute renal transplant rejection and of chronic cyclosporine nephropathy.2. The study in vitro showed that EMP can significantly promote the secretion of TNF-αand MCP-1, and up-regulate the expresstion of HLA-DR and CCR2 of monocyte-macrophage cells. It shows that EMP may participate in the injury of renal in CCN models by promoting the infiltration and activation of monocyte-macrophage cells.