Objective To explore the effect of VitC dialysate HDF on the oxidative stress and endothelial function. 　Methods Ten stable maintenance hemodialysis patients were enrolled in the research. The efficency of VitC dialysate HDF was compared with the regular HDF in prospective, self-cross way. Their plasma levels of total ascorbic acid (TAA), atocopherol, advanced oxidative protein products(AOPP) and malondialdehydea(MDA) were detected pre- and post-treatment of regular HDF or VitC dialysate HDF. The flow dependent vascular dilation (FDD) was measured by high-resolution B-mode ultrasonograph. The pre- and post-treatment serum was incubated with HUVEC, and then the levels of sICAM-1 and MCP-1 in the supernate were tested respectively.　 Results After regular HDF, the plasma levels of TAA and α-tocopherol decreased significantly[（39.78±21.52) vs. (21.51±13.88) μmol/L, (7.77±2.33) vs. (5.28±1.87) μmol/L, P<0.01], except for AOPP, MDA. After treated with VitC dialysate HDF, plasma TAA was significantly higher than before[(39.48±27.63） vs. (222.18±90.09） μmol/L， P<0.01], and α-tocopherol was significantly lower[（6.80±2.08） vs. （4.54±2.39） μmol/L, P<0.05]. FDD was markedly impaired after regular HDF（14.3±5.35 vs. 8.96±2.66, P<0.01; 67.82±32.32 vs. 33.34±15.23, P<0.05）, but no obvious change was found after VitC dialysate HDF（10.44±5.49 vs. 11.42±8.61, 60.29±38.15 vs. 70.53±56.05, P>0.05）. The post-treatment serum of regular HDF significantly stimulated the HUVEC secreting sICAM-1 and MCP-1, but such stimulation was not found by VitC dialysate HDF.　 Conclusions Regular HDF impairs the endothelial function probably for the VitC losing and high oxidative stress. The VitC dialysate HDF may be a good way to dissolve the problem partly.

Objective To elucidate the relationship between serum osteoprotegerin (OPG) levels and valvular calcification in chronic renal failure (CRF) patients. Methods The serum concentrations of OPG in 75 CRF patients and 10 age-matched controls were measured by ELISA. The presence of valvular calcification and its relationship with OPG levels were studied. Results Serum OPG levels in CRF patients [ND group（4.77±1.74） μg/L, PD group（5.22±1.57） μg/L, HD group（5.35±1.72） μg/L] were significantly higher than those in healthy controls [(2.04±0.57) μg/L, P<0.01]. The concentration of OPG was positively correlated with age（r=0.311, P<0.05） and C-reactive protein level（r=0.353, P<0.01）. Compared to CRF patients without valvular calcification, OPG level in CRF patients with valvular calcification was significantly elevated [(6.28±1.66) μg/L vs. (4.59±1.40) μg/L, P<0.01]. Logistic regression analysis showed that OPG level was an independent factor of valvular calcification(P<0.01). Conclusion Serum OPG level is correlated with the presence of valvular calcification in CRF patients.

Objective To study the molecular pathologic features of CD71 and correlation between IgA deposits and CD71 expression in IgA nephropathy(IgAN),and to investigate the role of CD71 in pathogenesis of IgAN. Methods According to clinical manifestations and pathological diagnosis, renal tissue samples of 120 cases were divided into IgA deposit nephritis (the primary group) 44 cases, non-IgAN with IgA deposit (the secondary group) 38 cases and no IgA deposit nephritis (the control group ) 38 cases. Immunofluorescence double-staining with TRITC-conjugated anti CD71 antibody and FITC-conjugated anti-human IgA antibody was performed to illustrate the expression of CD71 and IgA respectively under confocal microscopy. Results The expression of CD71 in the IgAN tissues was consistent with the accumulation of IgA. Significant differences of the expression of CD71 and IgA in glomeruli were found between primary and secondary group. The expression levels of CD71 and IgA were much higher in the　primary group than those in secondary group. Moreover CD71 expression was not found in control group. IgA and CD71 were co-localized under confocal fluorescence microscopy. Conclusion CD71 is highly expressed in glomeruli of primary IgAN and co-localized with IgA, which may play an important role in immunopathogenesis of IgAN.

Objective To investigate whether vector-based short-hairpin RNA（shRNA）could efficiently inhibit the expression of NHE1 in rat mesangial cells（MC） and the effect on aldosterone-mediatd proliferation of fibronectin (FN) in rat mesangial cells. Methods The eukaryotic vector of shRNA with insert targeting on the sequence of Na/H exchanger-1 （NHE1） was successfully constructed and transfected into rat mesangial cells. MCs were cultured till day 4 after transfection and the results were compared with those of the control group and non-specific transfected group. The mRNA of NHE1 were reverse transcribed and quantified by real-time PCR. Protein expression was assessed by Western blot. On day 4 after transfection with shRNA-NHE1, aldosterone (10-7 mol/L) was added to the medium, 24 hours later, the supernatant level of FN was examined by ELISA. Results After transfection of shRNA-NHE1, the mRNA expression of NHE1 decreased on day 1, and it decreased progressively on day 3 and day 4, while the suppression of NHE1 protein abundance did not appear until day 3. Its protein abundance was further decreased on day 4. ELISA showed the FN expression induced by aldosterone was increased compared with the control group [（51.78±1.15） vs.（17.74±1.38） μg/L, P < 0.05], and shRNA-NHE1 could inhibit this effect remarkably[FN(28.07±1.73) μg/L, P < 0.05]. Conclusion Vector-based shRNA is a potential tool to inhibit expression of NHE1 and shRNA-NHE1 can inhibit aldosterone-mediatd proliferation of FN in rat mesangial cells, which indicates that NHE1 may play an important role in aldosterone-mediated glomerular sclerosis.

Objective To investigate the role of NADPH oxidase in tubular epithelial-myofibroblast transition(EMT) induced by TGF-β1 in rat renal tubular epithelial cells. Methods Growth arrested and synchronized rat renal tubular epithelial cells (NRK-52E) were stimulated by 10 μg/L TGF-β1 for different time. Part of experimental cells were pretreated with DPI, an inhibitor of NADPH oxidase, for 1 h. The expression of NADPH oxidase subunits in cultured NRK-52E cells was messured by semi-quantitive RT-PCR. Intracellular ROS generation was visualized using H2DCF-DA by confocal microscope. The expression levels of α-SMA and E-cadherin were measured by RT-PCR, Western blot and immunocytochemistry, respectively. The mRNA and protein expression of PAI-1 and Col-Ⅰwere assessed by RT-PCR and Western blot respectively. Results TGF-β1 significantly upregulated the p67phox subunits mRNA expression, which was 2.43 folds and 3.59 folds of control group in 8 h and 24 h (P<0.01). TGF-β1 promoted the synthesis of cellular ROS, which was 2.5 folds of control group after 5 min stimulation (P<0.05). DPI could decrease the generation of ROS induced by TGF-β1(P<0.05). DPI effectively reversed TGF-β1-induced expression of α-SMA, E-cadherin, Col-Ⅰ, and PAI-1 in rat renal tubular epithelial cells. Conclusion TGF-β1 can significantly increase intracellular ROS and NADPH oxidase-derived ROS mediates TGF-β1-induced EMT in rat renal tubular epithelial cells.

Objective To explore the effect of decorin and TGF-β1 on rat mesangial cell (MsC) growth and its relative signal pathway. Methods Lipofectin-mediated method was used to transfect DCN vector into MsC. After the screen and identification of transfected MsC, DCN-containing supernatant was collected and it or/and TGF-β1(2 μg/L) was added into the culture medium of normal MsC. Flow cytometer was applied to detect the cell cycle. Western blot analysis was used to examine the expression of phospho-MAPKs, phospho-Smad2 and p21 protein. Inmmunoprecipation was applied to test the combination of DCN and TGF-β1 in supernatant of cultured MsC. Results Compared with normal MsC, the number of G2-M cell stimulated by DCN-containing supernatant or TGF-β1 decreased to 86.5% or 81%（P<0.05），and the expression of phospho-ERK1/2 and phospho-SAPK/JNK was obviously up-regulated(P<0.05), whereas the combination of both DCN and TGF-β1 could not obviously change above expression compared with control group. The protein level of p-Smad2 was increased with stimulation of TGF-β1 and p21 expression was increased with DCN stimulation, but the expression of both p-Smad2 and p21 did not obviously change after combination use of both DCN and TGF-β1 compared with control group. DCN and TGF-β1 in supernatant of cultured MsC could combine with each other. Conclusions TGF-β1 or DCN alone can suppress MsC proliferation and activate MAPKs signal pathway of MsC, but the effect of their combination is antagonistic. DCN can inhibit the activation of TGF-β1/ p-Smad2 signaling pathway. And up-regulation of p21 protein expression caused by DCN can be suppressed by TGF-β1. These all effects may be associated with the combination of DCN and TGF-β1.

Objective To elucidate the effects of aristolochic acid (AA) on human umbilical vein endothelial cells (HUVEC). Methods (1) The proliferation of HUVEC was determined by MTT method. (2) The cytotoxicity of AA-Na to HUVEC was determined by lactate dehydrogenase (LDH) release test. (3) HUVEC was stimulated with 5 mg/L AA-Na for different times. The mRNA and protein expression of TGF-β1, PAI-1 and TSP-1 were measured by RT-PCR and enzyme-linked immunosorbent assay (ELISA). Results (1) When the concentration of AA-Na was higher than 10 mg/L, the proliferation of HUVEC was significantly inhibited and LDH releasing from HUVEC was significantly increased (P<0.01)； (2) When HUVEC was treated with 5 mg/L AA-Na for 8~16 h, the mRNA expression of TGF-β1, PAI-1 and TSP-1 was significantly up-regulated to 1.53, 1.24 and 1.23 times respectively (P<0.05) compared with the control group； (3) When HUVEC was stimulated with 5 mg/L AA-Na, TGF-β1 was significantly increased at 24 h (P<0.01); PAI-1 and TSP-1 were also significantly enhanced at 16 h(P<0.01). Conclusions AA-Na of 5 mg/L can up-regulate mRNA and protein expression of TGF-β1, PAI-1 and TSP-1 of HUVEC, which may be related to the pathological changes of arterioles in chronic AA nephropathy.

Objective To investigate the effects of pravastatin on the MCP-1 expression induced by carboxymethyllysine modified bovine serum albumin (CML-BSA) in podocytes. Methods MCP-1 expression was detected by RT-PCR and ELISA. Dichloroflurescin-sensitive intracellular reactive oxygen species (ROS) generation was detected by confocal microscopy. Activations of extracellular signal-regulated kinase (ERK), NF-κB and Sp1 were studied using Western blotting and immunocytochemistry. Results CML-BSA could induce MCP-1 expression in a time- and dose-dependent manner in podocytes. Pre-treatment of podocytes with 0.1 and 1 mmol/L pravastatin inhibited CML-BSA-induced MCP-1 expression in gene and protein level. Podocytes could rapidly generate intracellular ROS after the treatment with CML-BSA. Pravastatin could not prevent ROS generation. Phosphorylated ERK was found in podocytes incubated with CML-BSA and was prevented by pravastatin in a dose-dependent manner. Both Western blotting and immunocytochemistry revealed that pre-treatment of podocyte with pravastatin prevented the NF-κB and Sp-1 translocation induced by CML-BSA. Conclusion Pravastatin inhibits MCP-1 expression induced by CML-BSA in podocytes via modulation of intracellular ERK, NF-κB and Sp1 signalling pathway.

Objective To study the influence of hyperphosphatemia on vascular calcification in chronic renal failure(CRF) rats. Methods Male Wistar rats (n=44) underwent 5/6 nephrectomy (n=24, model rats) or sham operation(n=20, control rats). From the 4th week after 5/6 nephrectomy, thirty nine rats were fed with high phosphorous (HP) diet [diet formular: phosphate (P) 1.2%, calcium (Ca) 1.6% and Vitamin D 1 IU/kg] or low phosphorous (LP) diet (diet formular: P 0.2%, Ca 0.5% and Vitamin D 1 IU/kg) for 10 weeks respectively. They were divided into four groups as follows: (1) CRF rats receiving HP diet (CHP group), (2) CRF rats receiving LP diet (CLP group), (3) control rats receiving HP (NHP group), (4) control rats receiving LP (NLP group). At the 4th week (baseline) and 14th week (end point), serum creatimine (Scr), serum Ca, serum P, serum 1,25(OH)2D3, intact parathyroid hormone (iPTH) and body weight were examined. At the 14th week, thoracic aorta was removed. Calcification were confirmed in the upper and lower part of aorta by Von Kossa staining and measurerment of calcium content. The middle part was frozen for measurement of core binding factor α-1 (Cbfα-1) mRNA by real-time PCR. Results At the 4th week (baseline), there were no significant differences of serum Ca, 1,25(OH)2D3, serum P,iPTH and body weight among 4 groups. Scr level in CRF rats was significantly higher than that in control rats (P<0.05). At the 14th week , there were no significant differences of Ca and body weight among 4 groups. 1,25（OH)2D3 level was slightly increased in CLP group compared to baseline(P=0.048), whereas no significant difference was found among other 3 groups. At the 14th week, Scr level in CRF rats was significantly higher than that in control rats (P<0.05). Serum P and iPTH levels increased significantly in CHP group compared with baseline (P<0.05).Vascular calcification was found in CHP group. with significant increase in calcium content of the aorta and Cbfα-1 expression as compared to any other groups（P<0.05）. There was a stronger relationship between serum P and calcium content than iPTH and calcium content (Beta:0.832>0.267). Serum P level had a linear positive correlation with calcium content and quantity of Cbfα-1 mRNA（r=0.672~0.73，P<0.05）. Conclusion Hyperphosphatemia is an important factor to influence vascular calcification in chronic renal failure rats, possibly through up-regulation of Cbfα-1.

Objective To establish a method culturing metanephric mesenchymal cells(MMCs) primarily derived from metanephric tissue of embryonic rats in vitro. Methods Pieces of rat metanephric tissue were explanted into plastic flask， and cells were grown in the tissue pieces. Cells were identified by inverted phase contrast microscope. Phenotypic protein examination was performed via flow cytometry and immunochemistry staining. Cells proliferation was measured by MTT and BrdU, and multi-direction differentiation was also induced. Results The cells showed shuttle shape with single nuclear in each one. Flow cytometry and immunochemistry staining revealed that significant expression of vimentin, fibronectin, CD29 and CD166 was positive over all of the cells and expression of CD31， CD34， CD45 and E-cadherin was negative in contrast， which confirmed the mesenchymal cell phenotype. Cultured cells could be propagated to 10 passages and remained the phenotype of mesenchymal cells. The cells could be induced to differentiate to adipocytes and osteocytes under special conditions. Conclusion Cultured mesenchymal cells from metanephric tissue of embryonic rats can be propagated and have strong proliferation ability and stability.