Objective To evaluate the efficacy and safety of leflunomide (LEF) in induction and maintenance therapy of type Ⅳ and type Ⅴ lupus nephritis. Methods A prospective single-center controlled clinical trial was conducted. Patients with biopsy-confirmed type Ⅳ and type Ⅴ lupus nephritis in one month were recruited. Patients were given either LEF (LEF oral 30 mg/d) or cyclophosphamide (CTX IV 1 g/month) combined with prednisolone (40～60 mg/d, tapered to 10 mg/d). After 6 months of induction therapy, 20 mg/d LEF and 1 g/3months CTX with prednisolone (5~10 mg/d) added respectively. Side effects were recorded and the efficacy and safety were evaluated. Results Forty patients were enrolled (19 in LEF group and 21 in CTX group), and 5 patients withdrew due to adverse events (2 in LEF group and 3 in CTX group) during induction therapy. The total response rate was 88.2％ in LEF group and 72.2％ in CTX group respectively. Complete remission rate was 52.9％ in LEF group and 44.4％ in CTX group after 6 months induction therapy respectively. There was no difference in the change of proteinuria, serum albumin, serum creatinine, C3 and SLEDAI between two groups. Eighteen response patients entered maintenance therapy (7 in LEF group, 11 in CTX group). LEF group was followed up for (18.6±6.5) months, and no patient was found relapse, and 3 patients received repeated renal biopsy, which showed the pathologic type was improved and the activity index decreased, but the chronic index increased slightly. CTX group was followed up for (25.1±9.6) months and 3 patients relapsed. Major adverse event in LEF group was infection, among which herpes zoster was the most common type. Major adverse events in CTX group were infection and amenorrhea . Conclusions LEF combined with steroid appears to be a safe and effective therapy within the induction and maintenance stage of the patients with type Ⅳ and Ⅴ LN. The long-term benefit of LEF needs to be confirmed by additional studies.

Objective To evaluate the relationship of C161T polymorphism in peroxisome proliferator-activated receptor γ (PPARγ) gene with glucocorticoid -induced osteoporosis(GIO) in Chinese population. Methods The C161T genotypes of PPARγ exon 6 were determined by PCR-RFLP in 208 healthy persons(group Ⅰ), 168 patients without GIO(group Ⅱ) and 104 patients of GIO(group Ⅲ) . Bone mineral density(BMD) at lumbar spine, femoral neck， trochanter and Ward’s triangle were measured by dual-energy X-ray absorptiometry(DEXA). Results Hardy-Weinberg equilibrium was evident for PPARγ polymorphism. PPARγ gene CC genotype frequency was lower in group Ⅲ as compared with groupⅠ. CT and TT genotype frequencies were higher in group Ⅲ as compared with groupⅠ. CC, CT and TT genotype frequencies were not significantly different among group Ⅱ, group Ⅱ+Ⅲand groupⅠ. BMD of lumbar spine in group Ⅱand gronp Ⅲpatients with CC genotype was lower as compared to those with CT+TT (P ＜ 0.05). In groupⅡ, the BMD of CC and CT+TT genotypes at lumbar spine area was (1.04±0.17) g/cm2 and (1.02±0.07) g/cm2 respectively. In groupⅢ, the BMD at lumbar spine area was (0.94±0.12) g/cm2, (0.83±0.08) g/cm2 of CC, CT+TT genotypes respectively. The significant difference was also presented after adjusted by age and BMI. Conclusions The frequency of the genotype is not significantly different between groupⅠand groupⅡ+Ⅲ, which indicates that genotype may not be related with the onset of glomerulonephritis. The frequency of the genotype is significantly different between group Ⅰ and group Ⅲ, which indicates that genotype may be related with the onset. Polymorphism of C161T in PPARγ gene may have an effect on the BMD of lumbar spine of the patients receiving glucocorticoid group (Ⅱ+Ⅲ).Allele C may be a protection factor of bone mass.

Objective To investigate the relative frequency and to elucidate the relationship between clinical and pathological features of primary focal segmental glomerulosclerosis(FSGS) in the south of China. Methods The data of 263 adult patients diagnosed as primary FSGS from 1988 to 2005, excluding secondary FSGS by renal biopsies, were reviewed and analyzed retrospectively. Results (1) The incidence of primary FSGS accounted for 7.02% of adult primary glomerular diseases confirmed by renal biopsies, and 6.33% of adult primary nephrotic syndrome(NS) within corresponding period. The predominant patients were young people. The patients presented various degrees of proteinuria, including nephrotic syndrome in 133 cases (about 50.6%). (2) The main pathological characteristics of the 263 FSGS cases were as follows: 48.4% of the patients had a glomerulosclerosis ratio more than 25%, 88.5% of glomerulosclerosis usually accompanied tubulointerstitial lesion, including severe lesions of graded 2 or 3 (about 25.2%). (3) Both glomerulosclerosis and tubulointerstitial lesion were correlated with renal insufficiency. The degree of glomerulosclerosis and tubulointerstitial lesion was negatively correlated with creatinine clearance, but positively with serum creatinine. A positive correlation was also found between glomerulosclerosis and tubulointerstitial lesion (P＜0.01). Tubulointerstitial lesion was a significant influencing factor of renal insufficiency. Conclusions Primary FSGS is one of the major pathological types of adult NS. Glomerulosclerosis and tubulointerstitial lesion, which contribute to renal insufficiency, progress severely when the patients are diagnosed as primary FSGS. Therefore early diagnosis and therapy is recommended to attenuate the disease and is one of the important topics for the nephrologists.

Objective To detect the urinary levels of monocyte chemoattractant protein 1 (MCP-1), β-catenin and cytokeratin 19(CK19) in patients with various renal diseases and to analyze the relationship among these molecular levels in urine and the clinical and pathological features of patients, especially the formation of crescent. Methods Urine specimens of 20 healthy subjects and 124 patients with various renal diseases were collected at the day of renal biopsy and one month after renal biopsy respectively. Urinary levels of MCP-1, β-catenin and CK19 were examined by enzyme linked immunosorbent assay (ELISA). Urinary creatinine, 24-hour urinary protein excretion and serum creatinine were detected at the same time. The indexes of cellular crescent and fibrous crescent were calculated according to the number and the size of crescents respectively. Results All the patients had significantly higher levels of urinary MCP-1, β-catenin and CK19 compared with healthy subjects （P < 0.01）.Levels of urinary MCP-1 and β-catenin were significantly higher in patients with cellular crescent than those in patients without crescent [median 247.54, range 22.17～2335.18 ng/mmol Cr vs median 113.55, range 1.75～1057.77 ng/mmol Cr; median 219.40, range 0.93～3827.50 ng/mmol Cr vs median 82.30，range 4.03～1632.58 ng/mmol Cr, P<0.01]. Urinary levels of MCP-1, β-catenin and CK19 were positively correlated to the indexes of cellular crescent ( r= 0.75，0.21，0.63， P< 0.01). Conclusions Urinary levels of MCP-1, CK19 and β-catenin can be detected By ELISA in healthy subjects and patients with various renal diseases. The levels of urinary MCP-1, β-catenin and CK19 are correlated to the indexes of cellular crescent in the renal biopsy specimens.

Objective To evaluate the efficacy of catheter-restricted filling using the gentamicin-heparin mixed solution in the prevention for catheter-related bacteremia (CRB). Methods Forty-three patients in our center were enrolled from Nov. 2004 to Mar. 2005 in this study, and were randomly assigned to receive either heparin lock solution (H group, heparin 45 g/L, 23 patients) or gentamicin-heparin mixed solution (GH group, gentamicin 4 g/L, heparin 45 g/L, 20 patients) as the catheter lock solution during the interdialytic period. The observative indicators included the incidence of CRB, the concentration of gentamicin in peripheral blood, the residual kidney function (RKF), the side effect of gentamicin, and the change of blood levels of CRP and IL-6. Results Mean CRB-free catheter survival days was 114.13±34.39 (31~200) in GH group and 127.40±32.85 (40~196) in H group. Accumulative catheter day of 2625 day was gained in GH group and 2548 in H group. CRB developed in two patients in the H group whereas none of the patients developed CRB in GH group, however the incidence of CRB between two groups was not significantly different. The trough and peak concentrations of gentamicin in GH group were (0.23±0.12) mg/L and (0.53±0.29) mg/L respectively after two weeks, and(0.26±0.15) mg/L and (0.67±0.32) mg/L after twelve weeks, and no statistical difference was found. The levels of CRP and IL-6 decreased in both groups. At the 16th week, the level of CRP decreased significantly compared to baseline in GH group (P < 0.05), but such difference was not found in H gruop. The RKF decreased in both groups, but the significant differences were not found between two groups. At the 16 weeks, the positive rate of gentamicin-fast E. Coli was 23.6% in GH group and 21.4% in H group respectively, with no significant difference. There were no other antibiotic-resistant bacteria or fungus found in stool culture. Conclusions The gentamicin-heparin mixed solution (4 g/L-45 g/L) is a good catheter lock regimen that has better safety and convenience, and its anti-coagulative efficacy is definite. It may be a beneficial means of reducing the CRB rate in hemodialysis patients with cuff-tunneled catheter.

Objective To investigate the effect of N-terminal fragment(LRR-WSC) fusion protein of polycystin-1 (PC-1NF) on collagen Ⅳ synthesis and degradation in rat glomerular mesangial cells and to explore its mechanism. Methods Rat glomerular mesangial cells were treated with different concentrations of PC-1NF in vitro. The mRNA expression of type Ⅳ collagen, metalloproteinase 2(MMP-2) and tissue inhibitor of metalloproteinase 1(TIMP-1) were detected by real-time fluorescence quantitative PCR. The concentration of type Ⅳ collagen was determined by ELISA. The expression level of c-fos and c-jun was examined by immunocytochemistry. The expression of PKC-α was detected by Western blot. Results After treatment with 4 mg/L PC-1NF for 48 hours, the mRNA level of hype IV collagen was down-regulated [（82±11） copies per million GAPDH] compared with that in the control [（103±16）copies per million GAPDH] （P<0.05）, so was the protein level of type Ⅳ collagen. The mRNA level of TIMP-1 was significantly decreased [（3040±370） copies per million GAPDH] compared with that in the control [（5530±480） copies per million GAPDH] （P<0.01）. However, the mRNA level of MMP-2[（2770±170） copies per million GAPDH] was significantly increased compared with that in the control [（1150±90） copies per million GAPDH] （P<0.01）. Meanwhile, PC-1 NF fusion protein treatment resulted in decreased protein levels of c-jun, c-fos and PKC-α. Conclusions PC-1 NF fusion protein may induce collagen Ⅳ degradation through increasing the ratio of MMP-2 to TIMP-1 in rat mesangial cells, inhibit c-jun and c-fos expression by modulating the PKC-α synthesis, and then decrease the production of collagen Ⅳ in rat glomerular mesanglal cells.

Objective To evaluate the effects of angiotensin II (Ang II) infusion on nephrin expression and podocyte apoptosis in vivo, and investigate the mechanism of proteinuria and glomerulosclerosis induced by Ang II infusion. Methods Thirty-six male Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang II (400 ng&#8226;kg-1&#8226;min-1) by osmotic mini-pump, or to be used as normal control. After the systolic blood pressure was measured, the 24 h urine sample was collected for analysis of proteinuria at day 7, 14, 21, 28. Animals were sacrificed at day 14, 28 respectively. Serum creatinine, renal pathological change, podocyte apoptosis were observed. Expression of nephrin mRNA and protein was examined by RT-PCR, Western blot, and immunofluorescence staining. Results (1) Ang II-infused rats developed significant hypertension associated with marked proteinuria. Significantly positive correlation was found between blood pressure and proteinuria (r = 0.80，P < 0.01). (2)At day 14, Ang II infusion induced the narrowing of slit diaphragm. At day 28, apoptotic podocytes were detected by electron microscopy and TUNEL assay [(2.7±1.6) apoptotic podocytes per glomerular cross section] in Ang II-infused rats. (3) Distribution of nephrin expression was changed from linear to granular pattern in Ang II-infused rats at day 14 and expression of nephrin mRNA and protein increased (P < 0.05). But at day 28, expression of nephrin mRNA and protein was decreased, and significantly negative correlation was found between the expression of nephrin and the number of apoptotic podocytes (r = 0.63，P < 0.01). Conclusion Change in nephrin expression may play a critical role in the pathogenesis of Ang II-induced podocyte apoptosis.

Objective To investigate the effects of advanced glycosylation end products (AGE) on DNA damage in cultured normal rat kidney fibroblasts (NRK-49F) cells, and the potential role of oxidative stress in AGEs-induced genotoxicity. Methods NRK-49F cells were treated with DMEM medium containing AGE-BSA (AGE-bovine serum albumin) at various concentrations (100, 200, 400, 800 mg/L) for 24 h. Cells were treated with serum-free DMEM medium or BSA at various concentration (100, 200, 400, 800 mg/L) for 24 h as control. To evaluate the potential role of oxidative stress in AGE-induced DNA damage, cells were preincubated with or without 10 mmol/L N-acetyl-l-cysteine (NAC) for 24 h and then were treated with AGE (400 mg/L) for another 24 h. Cell proliferation was measured by reduction of AlamarBlue. Single-cell gel electrophoresis (comet assay), a well-established method for quantifying DNA damage, was employed to analyze AGE-induced DNA damage. Results Compared with control medium and BSA, treatment with AGE caused significant inhibition of cell proliferation (P < 0.05)and increase of DNA damage (formation of comet) in a dose-dependent manner. Tail length of comet-formation caused by 200 mg/L AGE (P < 0.05) and 400 mg/L, 800 mg/L AGE (P < 0.01) was significantly longer than those of control group and BSA-treated group. Preincubation with antioxidant NAC attenuated AGE-induced DNA damage. Tail length of NAC pretreated group was significantly shorter than that of non-pretreated group (P < 0.05). However, treatment with BSA for 24 h did not show any increase in tail length (P > 0.05) and had no effect on cell proliferation (P > 0.05), compared with control medium. Conclusions AGE can induce DNA damage and inhibit cell proliferation in NRK-49F cells. Antioxidant can attenuate these effects. The enhanced oxidative stress may be one mechanism of underlying AGE-induced genotoxicity in chronic renal failure.

bjective To establish a method culturing myofibroblasts primarily from cortex of rat kidney. Methods Explanting the pieces of cortex of rat kidney into plastic flask, then cells grew out from the tissue pieces. Cells were identified by observation with inverted phase contrast microscope and electron microscope, as well as by phenotypic protein examination via immunochemistry staining and Western blot analysis. Results The cells showed shuttle shape with single nuclear in each one. Expression of vimentin and α-SMA was positive over all of the cells, which confirmed the myofibroblast phenotype. In contrast, expression of epithelial aminopeptidase P, cytokeratin, desmin, which were known as the markers for both endothelial cells and epithelial cells, was negative in these cells. Cultured cells could be propagated to 5 passage and remained the phenotype of myofibroblsts. Connective tissue growth factor(CTGF) and transforming growth factor β1(TGF-β1) could stimulate cell proliferation, but interferon-gamma had no effect on these cells. Conclusion Cultured myofibroblasts from the cortex of rat kidney can be propagated and have proliferative response to CTGF and TGF-β1 .

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.

Objective To elucidate the clinical and pathologic characteristics of IgA nephropathy associated with ankylosing spondylitis（AS）. Methods Clinical and pathologic data of 10 patients suffered from IgA nephropathy associated with AS were reviewed. They were admitted to our hospital from 1997 to 2006 and diagnosed by renal biopsy. Results The average age of nine male and one female patients was (28.6±6.8) years old. Four patients presented with asymptomatic hematuria, and six with hematuria and proteinuria. All the patients suffered from microhematuria and two from macrohematuria. The average 24-hour proteinuria was （1.56 ±1.53） g. Two had hypertension. Serum creatinine levels of all the cases were normal. Light microscopy examination showed that eight patients were mild mesangial proliferation with Lee’s classification grade Ⅰ or grade Ⅱ, and the other two were moderate to severe mesangial proliferation with grade Ⅲ or grade Ⅵ. Conclusion Mild mesangioproliferative IgA glomerulonephritis may be the major morphological pattern in AS patients with renal involvement.

Objective To investigate the clinicopathological characteristics and outcome of two subtypes of class Ⅳ lupus nephritis (LN). Methods Seventy-eight patients with class Ⅳ LN proven by renal biopsy were classified into two groups according to the 2003 ISN/RPS modified classification of LN. Class Ⅳ-G (56 patients) referred to involvement in more than 50% of glomerular capillary surface area. Class Ⅳ-S(22 patients) referred to involvement in less than 50% of glomerular capillary surface area. The clinical manifestations, pathology and therapeutic efficacy were compared between these two groups. Results There were no significant differences of age, gender, course of disease, mean arterial pressure, haemoglobin, C4, serum creatinine between two groups. Greater 24 hour urine protein [（4.34±2.67） vs. (3.52±3.46) g, P<0.05）]and lower C3 level [（0.41±0.17） vs. (0.46±0.16) g/L, P<0.05）]were observed in Ⅳ-G group. Systemic lupus erythematosus disease activity index (SLEDAI) was higher in Ⅳ-G group（14.39±4.07 vs. 11.53±4.15, P<0.05）.The immunohistochemical staining revealed that immune complex deposit was higher in Ⅳ-G group than that in Ⅳ-S group (2.11±0.54 vs. 0.35±0.39, P<0.01). The infiltration of mononuclear macrophage was more common in Ⅳ-G group (1.75±0.87 vs. 0.86±0.76, P<0.05), whereas fibrinoid necrosis was more frequent in Ⅳ-S group（11.66±17.26 vs. 13.61±20.01, P<0.05）. The higher active index （9.56±3.17 vs. 6.59±2.12, P<0.01）and lower chronic index ( 4.93±1.59 vs. 5.88±2.15, P<0.05） in Ⅳ-G group were detected. A total of 49 patients (Ⅳ-S 14, Ⅳ-G 35) were followed up for more than 1 year and received immune suppressive agents. There were no significant differences between two groups in the changes of proteinuria, complement, SLEDAI scores and serum creatinine. Conclusions There are some clinicopathological differences between Ⅳ-S and Ⅳ-G patients. The prognostic distinction between them remains to be proven.

Objective To investigate the pathogenesis of lipoprotein glomerulopathy (LPG) by analyzing the apolipoprotein E (apoE) gene of members in a family including four LPG patients and one asymptomatic carrier. Methods Laboratory tests for urinalysis, serum creatinine, plasma lipid and lipoprotein were performed on all the members of above family. Genomic DNA was extracted from peripheral leukocytes. The fragment of the apoE gene covering exon 4 was amplified by PCR. The gel-purified PCR products were directly sequenced and subcloned into the pMD 18-T vector. Recombinant plasmids were sequenced to investigate both alleles of patients. Results A nucleotide G to C point mutation in exon 4 of the apoE gene was confirmed in each patient and one asymptomatic member. This missense mutation encoded a new apoE variant that amino acid substitution of the proline residue for arginine residue at position 150 of apoE. Those carriers were all heterozygous of this novel point mutation apoE(arginine150 proline) with elevated plasma concentrations of apoE. Conclusions A new variant of apoE, apoE(arginine150 proline), has been identified in a family with 3 generations, including four LPG patients and one asymptomatic carrier. Study of this variant characteristics may be important for understanding the pathogenesis of LPG.

Objective To investigate the effects of dietary salt intake on the expression of cyclooxygenase 2(COX2) in renal medulla of spontaneously hypertensive rats (SHR) and normaltensive Wistar-Kyoto rats (WKY). Methods Rats at age of 6 weeks were randomly assigned to the following groups (n=6): WKY rats were fed with low salt diet containing 0.04% NaCl (WKY-LS), WKY with high salt diet containing 8% NaCl (WKY-HS), and SHR with low salt diet (SHR-LS), SHR with high salt diet (SHR-HS). The blood pressure (BP) was determined by automatic sphygmomanometer at the tail artery. The urine volume（UV） and urinary sodium excretion（UNa） were also measured. Urinary level of 6-keto prostaglandin F1α（6k-PGF1α） was detected by radioimmunity. Protein expressive level of COX2 in the renal medulla was assessed by immunohistochemistry and Western blot. Results The average baseline BP of SHR groups, measured 1 day before the start of the salt diet, was significantly higher than that of WKY groups（P<0.05）. After 3 days of different salt diet, BP of WKY-LS, WKY-HS and SHR-LS did not change obviously (P>0.05), while BP of SHR-HS group was significantly enhanced compared to basal level (P<0.05）. Both UV and UNa of SHR groups were significantly lower compared with those of WKY groups regardless of normal, low or high salt diet provided（P<0.05）. Levels of UV and UNa significantly increased in all the groups treated with high salt diet and decreased in those allotted low salt food, compared to baseline levels（P<0.05）. Urinary levels of COX2 metabolite 6k-PGF1α in both SHR and WKY groups responded similarly to dietary salt intake. Groups of high salt diet exerting an obvious increase and groups of low salt diet exhibiting a drop（P<0.05） with no variance were found between SHR and WKY having the same salt-loading（P>0.05）. Renal COX2 staining was mainly localized in medullary interstitial cells by immunohistochemistry. High salt diet for 1 week increased enormously renal medulla COX2 expression equally in both SHR and WKY groups, while low salt diet had no effects. The same tendency was further confirmed by Western blot. Conclusions High salt dietary induces further elevation of blood pressure and sodium retention in SHR group compared with WKY group. The expression of COX2 in the renal medulla is significantly enhanced in all rats fed with high salt diet and there is no significant difference between SHR and WKY. Renal medullary COX2 may perform normal expression and be involved in the regulation of water and sodium excretion in SHR, but take no part in the occurrence of hypertension.