Comparative Proteome Analysis Of Transplant Kidney Glomerulus In Rats

Objective: To analysis the alteration of protein expression patterns from rat syngeneic controls and allotransplantations transplant kidney glomerulus by proteomics technique, search and identify differential expression proteins as biomarkers which are related with acute renal rejection.Methods: 1. Optimization and establishment of the model of rat orthotopic kidney transplantation as Fisher's method; 2. Divided experimental animals into two groups: (1) Syngeneic controls (10 animals): Sprague-Dawely(SD)(♂)-to- Sprague-Dawely(SD)(♂); (2) Allotransplantation groups(10 animals): SD(♂)-to-Wistar(♂). Parallel groups of allo- and syn- transplanted rats were sacrificed on day 3 and 5 post-transplantation, at which points grafts were collected. Grafts were used for studying pathology of kidney transplantation and comparative proteome analysis with transplant kidney glomerulus in rats; 3. Acute rejection was diagnosed according to the Banff97 classification; 4. Identify differential expression proteins from rat syngeneic controls and allotransplantations transplant kidney glomerulus. Separate and identify differential expression proteins using 2-DE combine with MS. Namely, the proteins of two groups'glomerulus on day 3 post-transplantation were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE), then Coomassie staining, image analysis by PDQuest7.0 analysis software and then ten differential expression protein spots were digested by trypsin in-gel. The differential expression proteins'PMF were analyzed and then identified by matrix-assisted laser desorption/ionization time of fight mass spectrometry (MALDI-TOF-MS), searched against the NCBInr database using Mascot software; 5. Validate two of these differential expression proteins by using immunohistochemical staining, analysis the correlation between the pathology of AR and the differential expression proteins on day 3 and day 5 post-transplantation; 6. Analyze these both proteins which were soluble in the serum by enzyme-linked immunosorbent assay (ELISA).Results: 1. Optimization and establishment a model of rat orthotopic kidney transplantation. The time of donor kidney anastomosis is 40±10 min, the average time of operation is 110±10 min. 2. As examined from tissue morphology, the structure of glomerulus and tubules in syn-groups appeared normal basically, occasionally with sporadic matrix inflammatory cells infiltration on day 3 post-transplantation. There were no more inflammatory cells infiltration on day 5 post-transplantation. However, there were lots of inflammatory cells infiltration in periglomerular and around small artery, scattered in tubules matrix and the number of cells in glomerulus increased in allo-groups on day 3 post-transplantation; lots of inflammatory cells infiltrated into glomerulus along saccule, the endothelial cells of small artery shed, hemorrhage, thrombosis and small artery walls fibrinoid necrosis, lots of inflammatory cells infiltrated into renal interstitium widely, renal tubular cell degeneration, necrosis on day 5 post-transplantation. 3. Comparative proteome analysis of transplant kidney glomerulus in rats: (1) The results showed that well-resolved, reproducible 2-DE patterns of rat transplant kidney glomerulus(17cm IPG pH 3-10) of syn- and allo-groups on day 3 post-transplantation; (2) Twenty-four differential expression proteins were screened by analysising the electrophoretic maps of syn- and allo-groups, in which 6 spots were down-regulated and 18 spots were up-regulated; (3) Ten differential expression proteins were identified by PMF, obtained eight differential proteins'PMF, and found five up-regulated proteins: voltage-dependent anion channel(VDAC), Cofilin, glyceraldehyde-3-phosphate dehydrofenase(GADPH), CD44 and heat shock protein 90(HSP90) by bioinformatics. 4. The expression levels of CD44 and HSP90 in grafts: the results of immunohistochemical staining showed that CD44 expression in the syn-groups was negative in blood vessel endothelium, little expression in epithelial cells of Bowman's capsule wall, tubule and matrix. CD44 expression in the allo-groups was positive, widely and strongly located in glomerulus, small vessel endothelium, renal tubule of renal coetex and medulla, also renal matrix; HSP90 was present in the endothelium of the glomerular capillaries and the epithelium of renal tubules weakly in the syn-groups. The strongest signals were seen in the glomeruli HSP90 staining in the allo-groups. It seemed that they were localized predominanting in the endothelial cells of the glomerular capillaries and glomerlus, significant correlation differences was observed between the Banff scores for acute rejection and HSP90 staining of grafts; 5. The level of CD44 and HSP90 in serum: the results of ELISA showed that the level of CD44 and HSP90 in allo-groups increased obviously compared to syn-groups on day 3 and day 5 post-transplantation(P<0.05), but there were no statistical significance between the level of CD44 in allo-groups on day 3 and day 5 post-transplantation(P>0.05); However, statistical significance was observed between the level of HSP90 in allo-groups on day 3 and day 5 post-transplantation(P<0.05).Conclusion: Twenty-four differentially expressed proteins were screened, in which 6 spots were down-regulated and 18 spots were up-regulated. The differential expression proteins detected by PMF and searched against the NCBInr database using Mascot software, found 5 acute rejection associated proteins. In which CD44 and HSP90 strongly expressed in glomerulus at the early stage of acute rejection(3d post-transplantation), and significant correlation differences were observed between the Banff scores for acute rejection and HSP90 staining of grafts, the level of CD44 and HSP90 in serum of allo-groups'increased obviously compared to syn-groups. Therefore, CD44 and HSP90 could be the candidate biomarkers of AR. Our research provide theoretical and expremental proofs for CD44 and HSP90 as using for dianogsis or therapy of AR.