Objective:To study the mechanism of immune tolerance induce by protal venous injection of donor spleen cells on the dog model of renal transplantation.Methods:The donor spleen cells were injected through the protal vein during operation,one week later,renal transplantation was performed.IL-2 and IL-6 were studied by method of ELISA.Results:Level of IL-2 and IL-6 in protal venous group and cyclosporin group was higher than that of control group.There were no difference between protal venous group and cyclosporin group.Conclusion:Immune tolerance could be produced by protal venous injection of donor spleen cells.

Objective To investigate the HLA antibody incidence and type renal recipients with different age, and to study the echaracteristics in different age patients, for clinical reference to forecast renal rejection in different age patients. Methods With serum dated from January 2006 to June 2008, patients were classified into three groups: young group, with age below 35 years; middle age group, with age from 36 to 50 years; and old group, with age above 50 years. Penel reactive antibody (PRA) were detected using ELISA. Results Pretransplant HLA antibody incidences in the young, middle age, old group were 18.18%, 23.00% and 6.19%, respectively. In young group, HLA antibody incidences were 5.59% and 8.51% in male and female respectively. In middle age group, they were 21.30% and 25.38% in male and female respectively. In old group, they were 11.36% and 25.00% respectively. HLA Ⅰ and HLA Ⅰ + Ⅱ antibodies were mainly found in all the three groups in pretansplant. Conclusion HLA Ⅰ and HLA Ⅰ + Ⅱ antibodies were mainly found in pretransplant. Antibody incidence was higher in patients who had more than once renal transplant than that in transfusion and pregnancy female. Antibody incidence is higher in female than that in male.

Objective To analyze the sensitized factors in urinemia patients who waiting for renal transplantation.Methods 2429 patients with urinemia from April 2002 to December 2008 were subjected to the detection of panel reactive antibody, and classified into 5 groups according to their clinical data:(A) no history disease group (n = 1097) who never experienced transfusion, pregnancy and transplantation; (B) Transfusion group (n = 361) who received transfusion more than 200 ml; (C) Pregnancy group (n = 481) who experienced pregnancy; (D) Transfusion+ pregnancy group (n= 294) who experienced both pregnancy and transfusion; (E) Re-transplantation group (n = 196) who experienced failed transplantation before, and waited for the second renal transplantation.Results All the males in group A were negative for PRA, and females were weakly positive for HLA Ⅱ antibody.The incidence of PRA production in group B was 15.24 % (55/361).Thirty-nine patients were positive for PRA in group C with the incidence being 8.11 % (39/481).The PRA positive rate in groups D and E was 30.61 % (90/294) and 70.92 % (139/196) respectively.PRA intensity was more than 60 % in 72 patients in group E.Conclusion Transfusion and pregnancy caused lower incidence of PRA positive rate.The incidence was much higher in transfusion + pregnancy patients than that in patients with transfusion or pregnancy alone.Graft caused the higher incidence of PRA than by transfusion and pregnancy.

Objective To investigate the effect of sorafenib in ameliorating renal fibrosis and its possible mechanisms.Methods Rats were subjected to unilateral ureteral obstruction (UUO ) and intragastrically administered sorafenib.NRK-52E cells were treated with transforming growth factor-β1 (TGF-β1)and sorafenib. HE staining was used to visualize renal fibrosis.α-SMA and E-cadherin expressions in kidney tissue and NRK-52E cells were performed using immunofluorescence.The cell cycle of NRK-52E cells was determined by flow cytometry analysis.Smad3 and p-Smad3 protein expressions in NRK-52E cells were detected by Western blot analysis. Results HE staining showed that kidney interstitial fibrosis,tubular atrophy,and inflammatory cell infiltration in the sorafenib-treated UUO groups were significantly decreased compared with the vehicle-treated UUO group (P<0.05).Compared with those in UUO and TGF-β-stimulated NRK-52E groups,the expression of a-SMA decreased but E-cadherin expression increased in the UUO kidneys and NRK-52E cells of the sorafenib-treated groups (P<0.05).After 24 h stimulation with TGF-β1 5 ng/mL,the number of cell cycles arrested in G0/G1 phase was significantly increased and the number of cells that entered G2 ,S phase decreased (P<0 .0 5 ).Compared with that in TGF-β-stimulated NRK-52E groups, p-Smad3 decreased in the sorafenib-treated groups (P<0.05). Conclusion Our results suggest that sorafenib may be useful for the treatment of renal fibrosis through suppressing TGF-β/Smad3 signaling.

Objective To evaluate the effect of silver needle injection therapy on rat with sports muscle injury. Methods Twenty-one healthy male Wistar rats were randomly divided into the injury group (n = 3),the silver needle group (n=12) and the control group (n=3). The expressions of bFGF and GDNF in gastrocnemius muscle tendon junction were detected on 7 d ,14 d and 28 d post-injury. Results No significant difference in the appearance of the injured tissue was found in both two groups on 7 d post-injury. The appearance of the injured tissue was better in the silver needle group than that in the control group on 14 d and 28 d post-injury. The tissue was almost normal in the therapy group on 28 d post-injury; The expression of bFGF in the therapy group was higher than that in the injury control group on 7 d and 14 d post-injury (P < 0.01). The expression of bFGF markedly decreased in the therapy group compared with the control group (P < 0.01) on 28 d post-injury. The expression of GDNF in the therapy group was higher than that in the injury control group on 7 d ,14 d and 28 d post-injury (P<0.01). Conclusion The silver needle injection therapy has the therapeutic effect on sports muscle injury reparation, which can increase the expression of bFGF and GDNF efficiently.

Objective To investigate whether the effect of transient high glucose on inflammatory factors expression could be continuous in rat glomerular mesangial cell,and its relation with histone methylation modification.Methods Rat glomerular mesangial cells (HBZY-l) were divided into three groups:the high glucose group (25.0 mmol/L glucose),the hypertonic group (MA,5.5 mmol/L glucose+ 19.5 mmol/L mannitol) and the normal-glucose control group (5.5 mmol/L glucose),which were cultured for 24 h respectively.All 3 groups were then changed with normal-glucose medium to culture for 24 h,48 h and 72 h.Their protein,mRNA and supernatant were harvested.The protein expressions of mono-methylation of H3 lysine 4 (H3K4mel) was measured by Western blotting,and the mRNA expressions of NF-κB subunit p65 and set7/9 were determined by real timequantitative PCR.The expression of monocyte chemoattractant protein 1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) were detected by enzyme-linked immunosorbent assay.Results (1)Compared with those in normal control group,the expressions of H3K4mel protein and set7/9 mRNA were first up-regulated in high glucose group,then gradually down-regulated in the following 48 h normal-glucose medium (as compared with those at 0 h,all P ＜ 0.05).At 72 h there was no statistic difference between high glucose group and normal control group (all P ＞ 0.05).(2) Compared with those in normal control group,the up-regulated p65 mRNA,VCAM-1 and MCP-1 sustained at least for 72 h in high glucose group.Conclusions Transient high glucose can induce persistent inflammatory factors expression in rat glomerular mesangial cells,which may via histone modification.

Objective To explore the effect of transforming growth factor β1 (TGF-β1) on epigenetic histone lysine acetylation in the plasminogen activator inhibitor 1 (PAI-1) promoter and transcribe regions in glomerular mesangial cells (GMCs).Methods Chromatin immunoprecipitation assay and real-time quantitative PCR were used to detect Histone3K9 acetylation (H3K9Ac) in the PAI-1 promoter and transcribe regions induced by TGF-β1 and high glucose.Immunoprecipitation was also used to see the cooperation of Smad3,CBP and Sp1 proteins.Results In the four target regions of PAI-1 promoter,TGF-β1 treatment enhanced H3K9Ac at P1,P2 and P3 in GMCs (P ＜ 0.05),but no change was seen in the P4 region which was far from the transcription starting site.TGF-β1 obviously induced H3K9Ac in the T1 transcribe region of PAI-1 instead of T2 (P ＜ 0.05).High glucose increased PAI-1 mRNA expression and H3K9Ac around P1 promoter region (P＜ 0.05).TGF-β1 neutralizing antibody abrogated high glucose-induced H3K9Ac at PAI-1 promoter (P ＜ 0.01).TGF-β1 treatment could recruit Smad3 and CBP protein binding to the PAI-1 promoter regions (P1,P2,P3),and induce their cooperation in GMCs,which were responsing to TGF-β1 associated H3K9Ac.Conclusion TGF-β1 can induce H3K9Ac in the promoter and transcribe regions of PAI-1,promote Smad3 recruition and cooperation with Sp1 and CBP,which are associated with PAI-1 gene's regulation in GMCs.

Objective To investigate the effect of 12-lipoxygenase(12-LO) on the p27Kip1 expression in diabetic glomeruli. Methods Mesangial cells were exposed to 12-LO product 12 (S)-HETE (10-7 mmol/L) with or without p38 MAPK (p38) inhibitor (SB203580, 1 μmol/L) for 24 hours. Rats fed with high fat diet received low dose streptozotoein (ST-Z, 35 mg/kg, IP injection) to develop type 2 diabetes and were divided into 2 groups: low dose STZ, low dose STZ+12-LO inhibitor cinnamyl-3,4-dihydroxy-α-cynanocinnamate (CDC, 8 mg/kg) treatment. Rats fed with regular chow were divided into two groups: controls, CDC treatment. The rats received injection of CDC or vehicle subcutaneously in the hind leg. CDC or vehicle injection was performed three times weekly on alternate days. All the rats were sacrificed after 4 weeks, Wild type and 12-LO knockout C57BL/6 mice were divided into 4 groups: wild type control, 12-LO knockout, STZ-induced wild type type 1 diabetes and STZ-induced 12-LO knockout type 1 diabetes. All the mice were sacrificed after 16 weeks. Urine, blood, kidney cortical tissue and isolated glomeruli by sieving method were collected at the end of study respectively. Western blot and immunohistochemistry for target protein were performed respectively. Results Inhibition of p38 activation could significantly reduce p27Kip1 expression induced by 12 (S)-HETE in mesangial cells (P<0.01). Increased glomerular volume, microalbuminuria, elevated glomeluli p38 activation, p27Kip1 expresssion in type 2 diabetic glomeruli was decreased after CDC treatment (P<0.01). Compared with wild type diabetic mice, glomerular p38 activation, p27Kip1 exprcsssion and extracellular matrix accumulation in the 12-LO knockout diabetic mice were significantly decreased (P <0.01, respectively). Conclusions 12-LO induces p27kipl expression via p38 pathway in diabetic glomeruli.

Objective To study the expression of immunoglobulin-like transcripts 3 (ⅡT3) and ILT4 in peripheral blood dendritic cells (DC) of kidney transplantation recipients and to analyze its significance in immunity hyporesponsiveness of transplantation. Methods Twenty kidney allograft recipients who were survived more than five years were recruited to two groups: renal function stable groups, chronic rejection groups, and 10 healthy volunteers served as a control group. The peripheral blood mononuclear cells (PBMC) were stimulated with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) and immature DC were obtained. The expression of ILT3 and ILT4 was detected by using flow cytometry. The level of HLA-G5 in serum was determined by using enzyme linked immunosorbent assay. Results ILT3 expression in renal function stable group was increased and decreased in chronic rejection groups as compared with control group (P＜0.05),but ILT4 expression had no significant difference among all groups. HLA-G5 in serum was significantly increased in renal function stable group as compared with other groups. Conclusion Expression of ILT3 and HLA-G was increased in the kidney transplantation recipients with stable renal function and long-term survival, suggesting that they may play an important role in inducing and maintaining periphery immune tolerance.

Objective To investigate the expression vibration of microRNA-503(miR-503) and its effect on target gene Bcl-2,caspase enzyme activity and apoptosis of human renal tubular epithelial cells (HK-2) induced by high glucose,and to clarify the pathogenesis of renal tubular injury induced by high glucose.Methods HK-2 cells were cultured in normal glucose group (NG),mannitol hypertonic control group (MA),and high glucose group (HG).The morphology of apoptotic cells was observed using inverted microscope.The expression of miR-503 was determined using realtime quantitative PCR.The apoptosis rate of HK-2 cells was detected by Annexin V-FITC double dye using flow cytometry instrument.The expression of Bcl-2 and cleaved caspase-9 were detected by Western blotting.Results In the high glucose and mannitol groups HK-2 cell,an obviously increased apoptotic rate was observed under inverted microscope compared with normal glucose group (P ＜ 0.05).MA and HG up-regulated miR-503 expression (P ＜ 0.01),down-regulated anti-apoptotic protein Bcl-2 expression (P ＜ 0.05) and up-regulated cleaved caspase-9 (P ＜ 0.05).Conclusions The expression of miR-503 increases in HK-2 cells cultured by high glucose and mannitol.MiR-503 promotes apoptosis of HK-2 cells via activating mitochondrial apoptotic pathways and enhancing cleaved caspase-9 for Bcl-2 insufficiency.The tubular toxicity of high glucose is partly due to osmotic pressure.The miR-503 may be involved in diabetic tubular injury and may be a new therapeutic target of DN.