Chinese Journal of Nephrology

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Glomerular expression of podocin in patients with nephrotic syndrome

ZHOU Wei;CHEN Nan;PAN Xiao-xia;ZHANG Wen;XU Yao-wen;WANG Wei-ming;WANG Zhao-hui

2005, 21(9): 502-505.

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Objective To determine the distribution and expression of podocin in glomerular podocytes among nephrotic syndrome(NS) patients with different pathology and response to steroid. Methods Twenty-one NS patients were examined. Among them, twelve patients presented focal segmental glomerulosclerosis (FSGS), including seven steroid-resistant or steroid-dependent patients, and five steroid-sensitive patients,five patients were diagnosed as minimal change disease (MCD) and four patients as membranous nephropathy(MN). Three normal renal specimens were set as control group. Immunofluorescence staining, confocal laser scanning microscopy and LSM-510 graphic system were used to detect the expression and distribution of podocin in podocytes. Type IV collagen alpha 3 chain was also stained as location comparison. Results (1)In control group, the distribution of podocin showed a linear pattern along glomerular basement membrane. MCD and MN revealed a similar pattern. Part of FSGS, however, showed dotted or short linear patterns. (2)The podocin expression in NS patients with FSGS was significantly lower than that in normal kidney(80.5±33.5 vs 138.4±38.1, P < 0.05). In steroid-resistant and steroid-dependent patients, the expression of podocin decreased significantly; however in steroid-sensitive patients, the decrease of podocin was not statistically significant. (3)There were no significant differences of podocin expression among MCD(112.1±47.6), MN(92.5±34.8) and control groups(P=0.4497,P=0.1570 respectively). (4)No significant differences of podocin expression were observed in different pathology with NS(P > 0.05). Conclusion The expression of podocin decreases significantly in NS patients with FSGS, especially in steroid-resistant and steroid-dependent FSGS patients. Part of FSGS patients shows abnormal distribution patterns. The expression of podocin may play a role in the sensitivity of steroid.

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Analysis of proteomic components of sera from patients with uremia by two dimensional electrophoresis and matrix assisted laser desorption/ionization time of flying mass spectrometry

WANG Jian-qing;DAI Yong;DENG An-guo;LIU Jian-jun;HE Jian-fan

2005, 21(9): 506-511.

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Objective To establish and optimize two-dimensional gel electrophoresis technical platform, and compare the different sera proteomic components between uremic patients and normals. Methods Immobiline pH gradients isoelectric focusing was used as 1D, and vertical SDS-PAGE as 2D. Some applications, such as sample preparation and volume of loading sample, choice of IPG gel, were improved. Sliver staining, ImageMaster 2D 5.0 analysis software, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS) and IPI human database searching were used to separate and indentify the proteome from uremic patients sera.Results Satisfactory 2DE patterns of the uremic serum proteins were obtained. Twenty-six protein spots were remarkably changed in uremic patients, and 20 protein spots were identified by MALDI-TOF-TOF-MS. Conclusions Good reproducibility could be obtained by applying immobilized pH gradient 2DE to seperate and identify the proteome in serum, which contributes to further study on uremia toxins pertaining to protein.

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Study on the inhibitory effect of decorin on rat mesangial cell growth and its signal transduction pathway

FENG Xiu-yan;ZHANG Zhi-gang;ZHAO Zhong-hua;CHEN Qi;GUO Mu-yi

2005, 21(9): 512-516.

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Objective To explore the inhibitory effect of decorin(DCN) on rat mesangial cell (MsC) growth and on the expression of mitogen-activated protein kinases (MAPKs) and p21 protein. 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 added into the culture medium of normal MsC, then flow cytometer was used to detect the cell cycle. Western blot analysis was used to explore the changes of MAPK and p21 protein. Immunofluorescence was adapted to detect the expression of p21 on cultured MsC. Results Compared with normal MsC, the number of G2-M cells treated with DCN-containing supernatant decreased to 35%(P < 0.05), while the expression of phospho-ERK1/2 and phospho-SAPK/JNK on MsC was enhanced significantly (P < 0.05). DCN antibodies suppressed the increasing of phospho-ERK1/2 and phospho-SAPK/JNK in a dose-dependent manner. Western blot analysis and immunofluorescence revealed that DCN-containing supernatant up-regulated the expression of both total and nuclear p21 protein on MsC. This enhanced expression was attenuated by DCN antibody . Inhibitors U0126 and circumin of ERK and SAPK/JNK pathway could inhibit total and nuclear p21 up-regulation caused by DCN-containing supernatant (P < 0.05), while the inhibitor SB203580 of p38 pathway had no similar effect on MsC.Conclusion DCN-mediated transduction possibly by signaling molecule ERK1/2 and SAPK/JNK and p21 protein can suppress the cultured mesangial cell growth.

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Effect of NADPH oxidase activity inhibitor apocynin on extracellular matrix metabolism in rat diabetic nephropathy

ENG Yan-qiang;JIANG Zong-pei;JI Yu-lian;YU Xue-qing;GAO Ling;PENG Wen-xing;DONG Xiu-qing

2005, 21(9): 517-521.

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Objective To study the expression pattern of NADPH oxidase subunit p22phox mRNA and the effect of its activity in extracellular matrix metabolism in kidneys of diabetic rats. Methods Streptozotocin-induced diabetic rats were randomly assigned to two groups: untreatment diabetic group(DM) studied for 12 weeks, NADPH oxidase activity inhibitor apocynin treatment group(DM+Apo, 0.2 g·kg^-1·d^-1) for 8 weeks. Kidney expression of p22phox mRNA of NADPH oxdiase was evaluated by RT-PCR. The expression of fibronectin in the kidneys was studied by immunohistochemistry. Kidney MMP-9 activity was detected by zymography. Results Compared with normal control group, the expression of p22phox mRNA in the kidneys of DM rats was significantly increased at 4 weeks, 6 weeks and 8 weeks (P < 0.05), respectively; but normalized to control level at 12 weeks. During the whole course, there was no statistic difference of p22phox mRNA expression between DM+Apo and DM. Compared with those of DM at 8 weeks, glomerular volume, glomerular fibronectin expression, glomerular ECM, serum creatinine, 24-hour protein excretion and kidney hypertrophy index decreased obviously(P < 0.05), while MMP-9 activity increased significantly(P<0.05) in DM+Apo, respectively.Conclusions NADPH oxidase up-expression in the kidneys of diabetic rats may play an important role in the development of diabetic nephropathy at early stage. Inhibiting of NADPH oxidase activity relieves ECM accumulation and it may be hopefully served as an useful strategy for prevention and treatment of diabetic nephropathy.

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Albumin-induced rat tubular cell apoptosis and MAPKs signal transduction

AI Xiao-xi;DING Guo-hua

2005, 21(9): 522-526.

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Objective To evaluate the role of albumin and the involvement of mitogen-activated protein kinases(MAPKs) in rat tubular cells apoptosis. Methods Rat tubular cells (NRK-52E) were incubated with various concentrations (10, 20, 30 mg/ml) of delipidated, endotoxin-free bovine serum albumin(BSA) for 6, 12, 18 and 24 h, respectively. The process of apoptosis was evaluated by fluorescence microscope, transmission electron microscope, scanning electron microscope, confocal laser scanning microscope (CLSM) and flow cytometry. To assess the roles of p38, and the activities of JNK and ERK in albumin-induced apoptosis, SB202190 (20 μmol/L, p38 inhibitor), SP600125 (10 μmol/L, JNK inhibitor) or PD98059 (20 μmol/L, ERK inhibitor) were added to the NRK-52E cells separately in the presence of albumin for 24 hours. Activities of p38, JNK and ERK were assessed by Western blot analyses. Results The albumin induced tubular cell apoptosis in a dose- and time-dependent manner. Albumin stimulated the expression of p38 and JNK, whereas it inhibited the expression of ERK. SB202190 and SP600125 ameliorated tubular cells apoptosis but PD98059 treatment enhanced their apoptosis. Conclusions Albumin induces tabular cell apoptosis in a time- and dose-dependent manner in vitro, transducted through activation of p38 and JNK, and inhibition of ERK.

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Location and expression of human uric acid transporter in HKC

HONG Quan;WU Di;CHEN Xiang-mei;HOU Kai;FENG Zhe;FU Bo;XU Guo-shuang;DING Rui

2005, 21(9): 527-533.

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Objective To investigate the location and expression of human uric acid transporter (hUAT)within the human renal tubule epithelial cell. Methods The full-length hUAT/green fluorescent protein (GFP) fusion plasmid was constructed(hUAT-GFP), then it was transfected into renal tubule epithelium and Xenopus laevis oocytes. Meanwhile,the GST-hUAT fusion recombinant expression plasmid was constructed and the multi-clonal antibodies against GST-hUAT fusion protein were prepared. The localization of hUAT was determined within Xenopus laevis oocytes and renal tubule epithelium using immunofluorescence, Western Blot, Northern Blot and confocal microscopy. Results Rabbit multi-clonal antibodies against GST- hUAT fusion protein were prepared successfully. The hUAT was a membrane protein, which mainly expressed on the plasma membrane of human renal tubular epithelium. hUAT mRNA expression level in human renal tubular epithelium was up-regulated significantly when incubated with 400 μmol/L uric acid( P < 0.05). Conclusion hUAT is not a typical membrane protein, and may locate in the membrane in a dimmer form, whose location and expression is mediated through a special transcription regulation mechanism.

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Expression and role of CD40/CD40L and B7-1/CD28 interaction in folic acid-induced nephropathy

LI Xiao-zhong;DAI Ji-hong;YUAN Hai-tao;ZHANG Xue-guang

2005, 21(9): 534-537.

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Objective To explored the role of CD40/CD40L and B7-1/CD28 interaction in the immunopathologic process of folic acid-induced nephropathy(FAN) and investigate the intervention effect of the anti-B7-1 monoclone antibody(B7-1mAb). Methods CD1 mice were administrated by i.p with FA only or co-treated with B7-1 mAb for the intervention study. Kidneys were harvested for immunohistochemical, Western blot assays and serum for renal function evaluation at day 1, 3, 5, 7,14 and 21. Results Continuous expression of CD40, B7-1 from day 1 to day 21 on the renal tubular epithelial cells was detected in this model. CD40 was up-regulated more than five times in the kidney at every test time point (P < 0.01 for all) by semi-quantified of Western blotting. CD28 and CD40L were also observed in the infiltration of immunocopetent cell in the tubulointersititial area. After 21 days observation, death rate decreased from 47.83% to 11.01%(P < 0.01), the renal tissue protection area increased from 7.45% to 66.51% (P < 0.01) and plasma BUN and Scr level decreased. Conclusions The expression of CD40/CD40L and B7-1/CD28 can be detected in FAN. CD40/CD40L and B7-1/CD28 participate in renal tubular epithelial cell injury, immunocompetent cell infiltration and renal tubulointerstitial fibrosis. The success of B7-1mAb intervention provides special immune therapeutic targets for renal fibrosis.

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Superior renoprotective effects of the combination of enalapril with mycophenolate mofetil and its mechanism in diabetic rats

QI Xiang-ming;WU Yong-gui;WU Guo-zhong;LIN Hui;QIAN Hao;HAO Li

2005, 21(9): 538-542.

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Objective To assess superior renoprotective effects of the combination of enalapril with mycophenolate mofetil(MMF) and its mechanism in diabetic rats. Methods Diabetes was induced by injection of streptozotocin after uninephrectomy. Rats were randomly separated into five groups: control, diabetes, diabetes treated with enalapril, diabetes treated with MMF, and diabetes treated with combined enalapril and MMF. Albumin excretion rate (AER), the level of malondialdehyde (MDA) and activities of antioxidant in renal tissue were determined, and renal tissue morphology was observed by light microscopy after 8 weeks. Expression of ED-1, ICAM-1 and TGF-β1 protein was examined by immunohistochemistry or Western blot. Results Diabetes was associated with a considerable increase in albumin excretion rate. Both enalapril and MMF retarded the increase in albuminuria, which was nearly completely abrogated by combination therapy. Glomerular volume in diabetic rats was attenuated by treatment with either enalapril or MMF and further reduced by the combination of the two. Increased tubulointerstitial injury index was not lowered by enalapril or MMF treatment but reduced by the combination therapy in both cases. Elevated malondialdehyde level and decreased activities of superoxide diamutase,catalase and glutathione peroxidase in renal tissue were remitted by enalapril or MMF and, more effectively, by combined enalapril with MMF. Renal overexpression of ICAM-1 was not retarded by enalapril but attenuated by MMF or combined enalapril with MMF. Combination therapy was associated with a superior suppression in diabetes-induced macrophage recruitment and overexpression of TGF-β1 compared to either monotherapy in renal tissue. Conclusion The combination of enalapril and MMF confers superiority over monotherapies on renoprotection, whose mechanism may be at least partly related with synergetic suppression on increased oxidative stress and macrophage recruitment as well as overexpression of TGF-β1 in renal tissue.

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Effect of telmisartan on the expression of integrin α3β1 in the glomeruli of diabetic rats

CHU Gui-li;JIA Ru-han;GAO Ping;SONG En-feng;CHEN Cheng;DING Guo-hua

2005, 21(9): 543-547.

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Objective To observe the effect of telmisartan on the expression of integrin α3β1 in the glomeruli of diabetic rats. Methods Twenty-four SD rats were divided into three groups:normal rat, diabetic rat, diabetic rats daily treated with telmisartan (3 mg·kg^-1·d^-1, a dosage that has no effects on blood pressure) for 6 weeks. Blood glucose, blood insulin, blood pressure, 24-hour proteinuria, serum creatinine and Ccr were measured. The expression of integrin α3β1 in glomeruli was determined by immunohistochemistry and Westernblot. The expression of TGF-β1mRNA was examined by RT-PCR. In addition, the renal tissue pathology was observed by light microscopy. Ultrastructure of the glomeruli was observed by electron microscope. Results (1) No significant differences of blood glucose, blood insulin and blood pressure were found between diabetic rats and telmisartan treated group. Compared to diabetic group, the levels of serum creatinine, blood urea nitrogen, 24-hour proteinuria in telmisartan treated group decreased significantly. Renal pathologic changes and ultrastructure changes of the glomeruli in telmisartan treated group were also improved.(2)The expression of integrin α3β1 mainly distributed along glomerular vessels. Compared to normal group, the expression of integrin α3β1 was decreased in glomeruli of diabetic rats. After 6-week treatment, telmisartan significantly up-regulated the expression of integrin α3β1 compared to the diabetic rats, whereas it significantly down-regulated the expression of TGF-β1mRNA (0.39±0.06 vs 0.48±0.04,P<0.05). Conclusion Telmisartan attenuates proteinuria, improves renal pathology and protects against renal function in early stage of diabetic nephropathy in rats, possibly through up-regulating the expression of integrin α3β1 and down-regulating the expression of TGF-β1.

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Study on the protein permeability in high-flux dialysis membranes

XU Xiao-qi;Siegfried Stiller;Helmut Mann;QIAN Jia-qi;Heinrich Melzer

2005, 21(9): 548-551.

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Objective To evaluate the protein permeability of different high-flux dialysis membranes. Methods The filtrate was sampled from the dialysate compartment of different dialysers during the routine dialysis therapy. SDS-PAGE analysis was performed with silver staining method according to the modification of Melzer and consecutive laser densitometry. Before sampling all the dialysis mode was changed to pure ultrafiltration. Results The protein pattern of filtrate from dialysis membranes was similar to that of the glomerular membrane containing IgG, transferrin, albumin, retinol binding protein and beta-2-microglobulin. Comparing different membranes there were considerable differences depending on cut-off, charge and adsorption capacity of the particular membrane. In all membranes, permeability of proteins decreased during one treatment session. Conclusion Protein permeability of high-flux dialysis membranes is similar to the glomerular membrane but modified according to pore-size, surface charge, adsorption and time on dialysis. In contrast to the glomerular membrane, in each of the investigated membranes protein permeability decreases during the session.

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Effect of high-dose furosemide on volume state and residual renal function of CAPD patients

ZHOU Zai-sheng;WANG Bi-fei;YE Qing;WU Su-hong;CUI Hui-min;TIAN Min

2005, 21(9): 552-555.

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Objective To explore the effect of high-dose of furosemide on volume state and residual renal function. Methods Thirty-three continuous ambulatory peritoneal dialysis(CAPD) patients were randomly divided into trial group(17 cases) and control group(16 cases). All patients underwent standard CAPD. Only the patients of trial group received orally 100 mg furosemide twice daily. The trial lasted for 9 months and related clinical data were collected. Results At onset of the trial, the major clinical features, laboratory measurements and peritoneal transport characteristics were matched well. In the 3rd, 6th and 9th month of the trial,the urine volumes of trial and control groups were (788±198)ml and(701±187)ml､(813±220)ml and(673±194)ml､(809±209)ml and(599±176)ml,which were significantly differecent. However there was no statistically differece in Ccr decline between two groups. Inferior vena cava diameter index(IVCDI) was not different between trial and control groups(13.82±1.21 vs 13.78±1.09, P > 0.05) at the beginning of the trial, but it was markedly smaller in trial group than that in control group(11.72±1.10 vs 12.65±1.16, P<0.05) after the trial.Left ventricular mass index(LVMI) was almost equal(115.4±27.2 vs 115.7±29.4, P > 0.05) to two groups before the trial, but when the trial finished, LVMI of the control group was significantly higher than that of trial group(140.0±32.6 vs 120.9±24.5, P < 0.05). Conclusion High-dose furosemide can increase urine volume of CAPD patients and make the overload volume be controlled easily, thus decreasing the cardiovascular complications, such as hypertension and left ventricular hypertrophy.

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临床研究

基础研究

透析与移植

临床研究

基础研究

透析与移植