| Acute rejection, a key point to the long-term survive of renal grafts, is the major reason for the early damage and long-term dysfunction of the grafts and chronic allograft nephropathy. General diagnosis of acute rejection after renal transplantation includes four aspects as follows: (1)clinical manifestation;(2)laboratory test, some nonspecific biochemical indicator such as serum creatinine and urea nitrogen; (3)imageolgy examination, for example, color Doppler ultrasonography; (4)pathological biopsy. The first three tests, which are not characteristic for diagnosis, are obviously hysteretic so that the precise diagnosis would be made after the allografts had already suffered from acute rejection and been severely damaged. Biopsy, although thought to be the golden rule for the acute rejection final diagnosis, is an invasive way which probably causes terrible complications. Owing to the limited location for acquiring the sample, the whole status of the allografts can not be detected. Thus, early diagnosis biomarkers of acute rejection, which are prospective, noninvasive and specific are emergency requested to guide the clinical medication for the sake of increasing the survival rate of grafts.Urine reflects the physiological function and can be conveniently and safely collected, which is a perfect sample for non-invasive diagnosis of renal disease. Organ transplantation rejection is a complicated, dynamic and multiple factor procedure, which can not be accurately diagnosed by traditional unitary index. Proteomics which is dynamic and various in different time and status can systemically reflect the essence and rule of biological phenomenon. Thus we took the urine of pre-, mid- and post-AR as sample; find a series of candidate protein which might be meaningful for the diagnosis of acute rejection via two-dimensional gel electrophoresis mass spectrometry and initially validated these proteins.Firstly, we optimized the ampholine electrophoresis procedure since the urine protein had high electrolytes by which the protein dots number in 2-DE map was increased from 476 to 564 and the boosting pressure procedure was simple in contrast to the bio-rad recommended protocol. Meanwhile, in order to minimize the influence of electrolytes on 2-DE, we constructed a new way to acquire urine protein, namely acetone tripartite precipitation, by which the protein dots number in 2-DE pattern was average 761 which was obviously more than average 521 by acetone one step precipitation and the shape of the dots were distinct and circular, not to mention the level strap was apparently reduced.Secondly, in order to eliminate the individual difference between the urine protein samples, we choose two victims who suffered AR postoperative and compared the 2-DE map on different time points, which was three days, two days and one day before the clinical diagnosed AR (-3d,-2d,-1d)and the 7th,14th ,21st day after clinical diagnosed AR. Then we applied image analysis software to quantitate the 2-DE map and from the comparison of the 2-DE pattern on three days and two days before and the 21st day after the clinical diagnosed AR, we picked up the protein which was three times changed (P﹤0.05)and had the similar diversity trend in the two victims and identified them via mass spectrum.A total of 16 differential protein spots were successfully identified by MALDI-TOF-MS. Analyzed by means of bioinformatics and protein function index, alpha-1-antichymotrypsin (AACT), Zn-Alpha-2-Glycoprotein (ZAP) and tumor rejection antigen gp96 (gp96) were considered as candidate proteins for the further verification via immunoblotting. Four groups were established: urine from both of AR patients (n=8, 3-5days before clinical definition of AR) and stable patients (n=10, 7days after transplantation) after renal transplantations, urine from normal people (n=2) and operative patients of other diseases (n=2, 3days after operation) as control. Judgment from the imprinting straps by macroscopic observation, the accuracy rate, sensibility and specificity of these three proteins were: AACT, 77.78%/100%/60%; ZAG, 77.78%/100%/60%; gp96, 83.33%/100/80%. In view of these unsatisfied standers, we argued that protein existed or not is improper diagnosis stander, and the notable change of protein expression is also significant for diagnosing the disease. Therefore, the quantitative change of the aim proteins were analyzed by LINGO8.0 software, the accuracy rate, sensibility and specificity of the proteins were increased to 94.4%/ 100%/90.00%; 100%/100% 100%/ and 88.9%/100%/90.00%, respectively. Due to the limitation by sample volume, the study of multi-center clinical trials should be further carried out.In summary, our data suggest that early diagnosis of kidney graft acute rejection based on supposed criteria of AACT, ZAG and gp96 could be the promising in future. |
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