Objective     To compare the difference of ELISA and LABScreen in detecting HLA antibodies and evaluate their effects on allograft rejection.  Methods     Consecutive patients undergoing kidney transplantion from November, 2008 to December, 2009 in the First Affiliated Hospital and the following up patients during the same period, with abnormal Scr who had completed kidney biopsies, were included in this study. Patients’HLA antibodies were detected by ELISA (Lambda antigen tray, LATTM) and LABScreen Mix Beads (LABScreen MIX, One Lambda, Canoga Park, CA, USA) or LABScreen Single antigen beads (LABScreenTM single antigen beads, One Lambda, Canoga Park, CA, USA). Patients’Scr were also detected at different time potints.  Results     There were 277 patietns included. Among them 145 (52.3%) cases were HLA antibody positive detected by LABScreen, which including 118 cases ELISA negative but LABScreen positive, and 27 cases both ELISA and LABScreen positive. No case was ELISA positive but LABScreen negative. Among 118 cases which were LABScreen positive but ELISA negative, 41 (34.7%) cases happened acute or chronic rejection. However, only 24 cases happened rejection in 132 double negative cases (18.2%, P＝0.003). There were 31% patients in rejection group while only 12.8% patients (P＝0.01) in non - rejection group whose HLA antibody fluorescence intensity detected by LABScreen single antigen beads still increased two weeks after transplantation.  Conclusion     LABScreen is more sensitive than ELISA in detecting HLA antibodies, and its result highly correlates with the incidence of allograft rejection.

Objective    To evaluate the prevalence of hyperuricemia in patients with IgA nephropathy and find out the risk factors of hyperuricemia, including clinical and pathological characteristics.  Methods    A retrospective study enrolled 2566 adult patients, who admitted to the First Affiliated Hospital, Sun Yat-sen University from 1996.01 to 2012.12 and diagnosed with biopsy- proven IgA nephropathy was conducted.  Results    Among 2566 IgA nephropathy patients, the prevalence of hyperuricaemia was 36.6%. Prevalence of hyperuricaemia for CKD stage 1, 2, 3, 4, 5 was 16.2%, 37.4%, 66.4%, 87.7% and 76.4%, respectively. Adjusting Logistic regression analysis showed male gender, progressive stages of CKD, increased percentage of global glomerulosclerosis were independent risk factors of IgA nephropathy; male gender, progressive stage of CKD, increased level of cholesterol, increased percentage of global glomerulosclerosis were independent risk factors for CKD stage 1 - 2 patients; progressive stages of CKD and increased percentage of global glomerulosclerosis were independent risk factors for CKD stage 3 - 5 patients. Conclusion    The prevalence of hyperuricemia in patients with IgA nephropathy was 36.6%, and identifying the risk factors associated with hyperuricaemia among different CKD stages of IgA nephropathy will be important to improve our understanding in intervention of this disease.

Objective    To study skeletal muscle atrophy and the change of autophagy in skeletal muscle of patients with chronic kidney disease.  Methods    Mean muscle cross sectional area, mRNA and protein expression of autophagy markers Bcl-2-adenovirus E1B interacting protein 3 (LC3B), Bcl - 2 - adenovirus E1B interacting protein 3 (Bnip3), Beclin - 1 were measured in rectus abdominis biopsies obtained from 22 consecutive patients with stage 5 CKD scheduled for peritoneal dialysis from 4 hospitals in Shanghai. Control biopsies were obtained from another 8 healthy subjects during elective surgery for adenomyosis and 6 subjects during elective surgery for abdominal wall hernias. Rectus abdominis muscles were obtained at the beginning of surgery. HE staining was performed and mean cross sectional area (CSA) was calculated. Electron microscopy was used to confirm the changes of autophagy. mRNA levels of LC3B, Beclin-1, Bnip3 were evaluated by RT-PCR and protein levels of those parameters were evaluated by Western blotting.  Results    Compared with control group, mean CSA of muscle fibers was decreased and the transcript levels of LC3B, Beclin-1, Bnip3 were up-regulated in CKD group. Similarly, protein levels of LC3BI，LC3B II，Beclin-1 and Bnip3 were increased in CKD group. Additionally, activation of autophagy was confirmed by the appearance of autophagosomes by electron microscopy.  Conclusion    Chronic kidney disease may cause skeletal muscle atrophy and lead to activation of autophagy, which may contribute to muscle atrophy.

Objective    To assess the effect and safety of lanthanum carbonate vs conventional phosphate binders for hyperphosphatemia in patients undergoing maintenance hemodialysis.  Methods    According to the collaborative search strategy, MEDLINE (1996 to 2012.12), EBCO (1996 to 2012.12)，the clinical control test database of Cochrane Library and Chinese Wanfang database (1996 to 2012.12) were searched. Related literature, whether Published or not and meeting summary included, were searched by hand. Quality assessment and data extraction were conducted by two independent investigators. Meta - analysis was conducted by RevMan 5.0. The following outcomes were assessed: serum phosphorus levels, serum iPTH levels, serum calcium levels and adverse events. Results were expressed as OR with 95% confidence interval for dichotomous outcomes and WMD with 95% confidence interval for continuous outcomes.  Results    A total of 10 reports were identified which met the inclusion criteria. The meta-analysis showed that the efficacy of treating hyperparathyroidism in hemodialysis patients was similar between lanthanum carbonate and conventional phosphate binders (WMD＝-0.06, 95% CI-0.27 to 0.15, P＝0.57) and the incidences of discontinuing due to adverse events were also similar. However, there were fewer hypercalcemic episodes and lower serum calcium levels in the lanthanum carbonate group compared to calcium-based phosphorus binders group.  Conclusion    Lanthanum carbonate is effective and well tolerated in treating hyperphosphatemia in hemodialysis patients with fewer hypercalcemia and lower serum calcium levels compared to calcium- based phosphate binders.

Objective    To investigate the effect of Cordyceps sinensis on renal fibrosis and its possible mechanism.  Methods    Thirty Sprague - Dawley rats were randomly divided into three groups: Sham operation group (Sham，n＝10), 5/6 subtotal nephrectomy group (SNx, n＝10), and 5/6 subtotal nephrectomy treated with Cordyceps sinensis group (CS, n＝10). Body weights were assessed and 24 - hour urine excretion was collected before and every four weeks after surgery. Rats were sacrificed at 12 weeks after surgery. Blood samples were taken for biochemical study, and kidney tissues were used for HE and Masson stains to assess histological changes. Immunohistochemical staining was used to detect the expression of transforming growth factor β1 (TGF-β1) and its receptors of type Ⅰ(TβRⅠ), type Ⅱ (TβRⅡ). Immunofluorescence was used to detect E-cadherin and α-SMA. The relative protein level of TGF-β1, TβRⅠ, TβRⅡ, p-Smad2/3, Smad7, E-cadherin, α-SMA were examined by Western blotting.  Results    CS group had higher body weights and lower urinary protein, BUN and Scr level compared with SNx group. Glomerulosclerosis index and tubulointerstitial injury score were significantly reduced in CS group compared with those in SNx group (all P＜0.05). The protein expressions of TGF-β1,TβRⅠ, TβRⅡ, p-Smad2/3 were decreased in CS group compared with those in SNx group (all P＜0.05). CS treatment up-regulated the expression of E-cadherin, Smad7 and down - regulated the expression of α - SMA compared with that in SNx group (all P＜0.05).  Conclusion    Cordyceps sinensis has inhibitory effect on renal fibrosis in 5/6 subtotal nephrectomy rat model, which might be related with the suppression of TGF-β1 signal pathway.

Objective     To investigate the effect of erythropoietin (EPO) on the expression of homing factors of remnant renal tissue from rats with chronic kidney failure (CRF).  Methods     The CRF model was established by a two stage 5/6 nephrectomy procedure in rats. Experimental rats were randomly divided into three groups: sham operation group, CRF model group, EPO treatment group (CRF rats treated with human recombinant EPO). CRF rats received EPO by hypodermic injection with 50 IU/kg three times a week for 6 weeks and then were sacrificed. Serum creatinine (Scr), blood urea nitrogen (BUN), urine protein and haematoglobin (Hb) were measured. The expression of EPO and its receptor (EPOR), homing factors and their receptors (SDF-1, CXCR4, Ang-1, Tie2, SCF, c-Kit) in remnant kidney tissue were detected by the methods of real - time PCR, Western blotting and immunohistochemistry.  Results     Compared with CRF model group, the expressions of homing factors and their receptors (SDF-1, CXCR4, Ang-1, Tie2, SCF, c-Kit ) in remnant kidney tissue were up- regulated by administration of EPO in treatment group (all P＜0.05). Meanwhile, the expressions of EPO and its receptor in remnant kidney tissue were also up- regulated by administration of EPO in treatment group (all P＜0.05). Moreover, the Scr, BUN and urine protein in EPO treatment group were lower than those in CRF model group (all P＜0.05). Instead, haematoglobin was higher than that in CRF model group (P＜0.05).  Conclusion    EPO can improve renal function in rats with chronic renal failure, maybe through activation of homing factors in remnant kidney tissue which are involved in repairing damaged kidney.

Objective    To investigate the effect of adrenomedullin on rat renal tubular epithelial cell line (NRK - 52E) apoptosis induced by hypoxia - reoxygenation (HR) injury and its mechanism.  Methods    NRK-52E cells were cultured and randomly allotted to the following 4 groups: control group, HR group, empty plasmid ＋ HR group, ADM ＋ HR group. NRK - 52E cells were transfected with pcDNA3.1 - myc - his B empty vector or pcDNA3.1 - ADM by transfection complex comprising optimal proportion of plasmid and Fugene HD reagents. Cells were counted by trypanblau and cell survival rate was computed. The concentration of lactate dehydrogenase (LDH) was detected by spectrophotometric method to evaluate cell vitality. The apoptotic rate of NRK-52E cells was measured by flow cytometry. The transfer efficiency was detected by immunocytochemistry. The mRNA expressions of ADM, Bax, Bcl-2 and Fas were determined by Semi-quantitative RT-PCR, and active caspase - 3, 8, 9 protein expression were examined by Western blotting.  Results    Compared with control group, the expression of ADM significantly increased in HR group (P＜0.05). The expression of ADM significantly increased in ADM＋ HR group than that in empty plasmid＋ HR group. Compared with control group, in HR group, the living cell counts and cell survival rate significantly decreased; the LDH concentration in media, apoptotic rate and the levels of Bax, Bcl-2, Bax/Bcl-2, Fas, active Caspase - 3, 8, 9 significantly increased (all P＜0.05). Compared with HR group, in ADM ＋ HR group, the living cell counts and cell survival rate significantly increased; the LDH concentration in media, the cell apoptotic rate and the levels of Bax, Bax/Bcl-2, Fas, active Caspase-3,8,9 significantly decreased, while Bcl - 2 was promoted (all P＜0.05). The above indexes had no differences between empty plasmid＋ HR group and HR group (all P＞0.05).  Conclusion    The increased expression of ADM can inhibit NRK-52E apoptosis induced by HR, and the mechanism might be achieved by inhibiting mitochondrial and death receptor-mediated apoptotic pathways.

Objective    To investigate the impact of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells (HASMCs).  Methods    Uremic serum was incubated at 37℃ for 3 days. Calcium phosphate crystals and uremic supernatant were isolated from uremic serum by ultracentrifugation. Scanning electron microscope (SEM) and energy dispersive X - ray spectroscopy (EDX) were performed for analysis of morphological and chemical characteristics of the crystals. HASMCs were treated in vitro with control medium, uremic serum - medium, calcium phosphate crystals - medium and uremic supernatant - medium. Calcification was visualized by Alizarin red staining. Calcium loads in cells were quantified by o - cresolphthalein complexone method. Gene expression of bone morphogenetic protein-2 (BMP-2), osteopontin (OPN) and core-binding factor α1 (Cbfα1), alkaline phosphate (ALP) and matrix gamma carboxyglutamic acid protein (MGP) were quantified by real - time PCR. Cbfα1, OPN and BMP - 2 protein levels were determined by Western blotting or ELISA.  Results    Calcium phosphate crystals which induced by uremic serum displayed laminated shapes containing crystallized needle- like projections and ranged from 30 - 500 nm, with Ca/P ratios of 1.41±0.05. Compared with the cells in control group, uremic serum induced HASMCs calcification，increased calcium loads (P＜0.05), up-regulated BMP-2, OPN , Cbfα1 mRNA and protein expression (all P＜0.01). Similar to uremic serum, calcium phosphate crystals also induced HASMCs calcification, increased calcium loads (P＜0.05), and up-regulated BMP-2, OPN , Cbfα1 mRNA and protein expression (all P＜0.01). However, there was no significant difference between HASMCs growing in uremic supernatant and control medium in calcium loads or the expression levels of these osteogenic proteins (P＞0.05).  Conclusions    Calcium phosphate crystals induced by uremic serum promote HASMCs calcification, which might be one of the mechanisms of uremic vascular calcification.

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.