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Impact of pamidronate disodium on the calcification of vascular smooth muscle cells induced by hyperphosphate

Lei BU ; Xuezhi ZHAO ; Xinyu LI ; Jian ZHANG

Chinese Journal of Nephrology.2010;26(12):920-923. doi:10.3760/cma.j.issn.1001-7097.2010.12.010

Objective To investigate the protective effect and mechanism of pamidronate disodium on calcification in rat vascular smooth muscle cells (RVSMCs) induced by hyperphosphate.Methods RVSMCs were placed in various culture mediam, including normal phosphate medium (Pi 1.4 mmol/L), high phosphate medium (Pi 4.5 mmol/L), different pamidronate disodium concentrations medium (Pi 4.5 mmol/L+pamidronate disodium 10-5, 10-6, 10-7 mmol/L). Calcium content and cell protein content were quantified by the O-cresolphthalein complexone method and BCA protein assay respectively. Calcification was visualized by yon Kossa staining, and cbfα-1,osteocalcin were quantified by Western blotting. Results After culture for 3 days, calcium content in high phosphate group were much higher than that in control group, and pamidronate disodium groups had a lower calcium content compared with high phosphate group (all P＜0.05).Calcium deposit in RVSMCs was greater in high phosphate group, while pamidronate disodium groups revealed obviously decreased deposit (all P＜0.05). Protein expression of cbfα-1 and osteocalcin in pamidronate disodium groups was much lower than that in high phosphate group.Conclusion Pamidronate disodium can protect RVSMCs from phosphate-induced calcification in vitro, which may be associated with the blockage of transformation of RVSMCs into osteoblast.

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Impact of vascular endothelial growth factor and basic fibroblast growth factor on culture of rat peripheral blood progenitor cells

Jianjiang ZHANG ; Liping ZHENG ; Hua WANG

Chinese Journal of Nephrology.2010;26(12):915-919. doi:10.3760/cma.j.issn.1001-7097.2010.12.009

Objective To observe the impact of different culture conditions on the growth of the endothelial progenitor cells(EPCs) from rat peripheral blood. Methods Mononuclear cells obtained from rat peripheral blood were isolated by using density gradient centrifugation. According to the culture medium added with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and the dishes were precoated with fibronectin or not, the mononuclear cells were divided into different groups and cultured under different conditions in vitro to compare the differences of the growth conditions. The different growth conditions were recorded and the result was calculated by statistics software. At last, the cells were identified by immunohistochemistry and immunofluorescence. Results The mononuclear cells from rat peripheral blood grew in the manner of keeping close to the wall in vitro. The cells and cell colonies cultured for 7 days hinted:under the same culture conditions, precoating FN was beneficial to the adherent proliferation of EPCs (t=4.43, P＜0.05; t=3.70, P＜0.05). Excluding the impact of fibronectin, growth factors could promote mononuclear cells differentiation to EPCs, indicating that growth factors enhanced proliferation of EPCs (t =-13.22, P＜0.01; t =-10.96, P＜0.01). Immunohistochemistry and immunofluorescence showed that, at day 4, 7, 10 of cells cultured, CD34 and CD133 expressions were increased gradually [(35.7±4.2)%, (60.1±3.8)%, (81.8±6.4)%; (3.2±0.9)%, (18.4±7.3)%,(32±3.8)%, respectively]; at day 14, both were decreased [(32.1±5.4)%, (1.9±2.7)%]; but Flk-1was increased at day 4, 7,10, 14 [(31.2±3.5)%, (40.6±5.3)%, (71.2±8.4)%, (81.5±4.1)%].Conclusions Fibronectin is conducive to adhesion and proliferation of EPCs. VEGF and bFGF play an important role in the differentiation of EPCs. The success culture of EPCs in vitro will provide a sufficient number of seed cells for its application in vascular tissue engineering, and offer new ideas for peripheral blood stem cell transplantation for the treatment of various diseases.

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Uric acid stimulates rat glomerular mesangial cells proliferation via activation of extracellular signal-regulated kinases

Hong XIANG ; Songming HUANG ; Quancheng FENG ; Yanggang YUAN ; Aihua ZHANG

Chinese Journal of Nephrology.2010;26(12):892-897. doi:10.3760/cma.j.issn.1001-7097.2010.12.004

Objective To explore the effect of uric acid (UA) on the rat glomerular mesangial cells (GMCs) and its possible mechanism in vitro. Methods Cultured rat GMCs were treated with various concentrations of UA (50 μmol/L, 100 μmol/L, 300 μmol/L) in the presence or absence of U0126, apocynin, rotenone. The incorporation of 3H-thymidine (3H-TdR) and cell counting were used to measure GMCs proliferation. GMCs cell-cycle was analyzed by flow cytometry. CyclinD1 and cyclin A2 expression were determined by real-time PCR and Western blotting analysis. ERK1/2 phosphorylation was detected by Western blotting. Reactive oxygen species (ROS) was measured by 2, 7-dichlorofluorescein diacetate (DCFDA) fluorescence. Results (1) Uric acid increased GMCs proliferation in dose-dependent manner compared with the control groups, as assessed by 3H-TdR incorporation and cell counting. GMCs proliferation induced by 300 μmol/L uric acid was increased by more than 1.5-fold assessed by both of the methods. (2) Uric acid decreased cell number in G1/G0 phase and increased cell number in S phase in dosedependent manner, as assessed by flow cytometry. (3) Uric acid iuduced cyclin D1 and cyclin A2 expression in dose-dependent manner. (4)Uric acid increased pospho-ERK1/2 in dose-dependent manner and ERK1/2 specific inhibitor U0126 could suppress uric acid-induced cell proliferation.The inhibition percentage of U0126 was 22% and 31% assassed by cell counting and 3H-TdR incorporation, respectively. (5) Uric acid increased ROS production in dose-dependent manner.NADPH oxidase inhibitor apocynin could also significantly inhibit uric acid-induced ROS production, ERK phosphorylation and cell proliferation. In contrast, rotenone had no effect on them.Conclusion Uric acid can stimulate rat GMCs proliferation, partially by the activation of ERK pathway via NADPH oxidase-derived ROS generation.

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Meta-analysis of impact of continuous renal replacement therapy dose on outcome of acute renal failure patients

Hualin QI ; Feng LIU ; Jun WANG ; Chen YU ; Haidong YAN

Chinese Journal of Nephrology.2010;26(12):880-886. doi:10.3760/cma.j.issn.1001-7097.2010.12.002

Objective To assess the effect of continuous renal-replacement therapy (CRRT) dose on the outcome of acute renal failure (ARF) patients with meta-analysis of randomized controlled trials (RCTs). Methods Studies were identified by systematic search of peer-reviewed publications in Medline, EMBASE and Cochrane library database through June 2010. All the RCTs that compared the incidence of clinical outcome such as mortality, need for chronic dialysis between standard and low dose CRRT were eligible. The pooled relative risk (RR) for clinical outcome was compiled using a random-effects model. Heterogeneity was evaluated by means of subgroup and sensitivity analysis. Results Six eligible studies were identified. By meta-analysis, standard dose CRRT was associated with non-significant 13% mortality risk reduction (RR 0.87, 95%CI 0.70-1.07, P=0.19)and 13% composite outcome risk reduction of chronic dialysis dependence and mortality (RR 0.87, 95%CI 0.69-1.09, P=0.21), but the trend toward increased chronic dialysis dependence risk among survivors (RR 1.43, 95%CI 0.94-2.18, P=0.09). The overall test for heterogeneity among cohort studies was significant (P=0.001, I2=76.2%). The risk of mortality was modality was significantly lower in some studies of which delivered dose was moer than 35 ml·kg-1·min-1,modality was continuous venous-venous hemofiltration (CVVH) and major cause was non-sepsis treated with standard dose CRRT. Conclusions Standard dose CRRT in patients with ARF does not improve survival, renal recovery and composite outcome, but decreases mortality in important subgroups including those with higher delivered dose, CVVH and non-sepsis.

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Systemic lupus erythematosus with secondary antiphospholipid syndrome: a retrospective clinicopathological analysis of 11 cases

Wenqing YU ; Yubing WEN ; Ruitong GAO ; Hang LI ; Xuemei LI

Chinese Journal of Nephrology.2010;26(12):875-879. doi:10.3760/cma.j.issn.1001-7097.2010.12.001

Objective To analyze the clinicopathological characteris tics of systemic lupus erythematosus (SLE) with secondary antiphospholid syndrome (APS) . Methods Data of 11 cases of SLE with secondary APS (SLE with APS) admitted to Peking Union Medical College Hospital from January 2000 to March 2010 were retrospectively analyzed. Kidney biopsy was performed on all of these patients. Differences of clinicopathology and outcomes between SLE with and without APS were compared. Results Renal involvement was found in all the SLE with APS patients. The prominent clinical manifestations included hypertension (54.5%), nephrotic level of proteinuria (24 h proteinuria ≥3.5 g)(72.7%) and renal insufficiency (45.5%). Diastolic blood pressure, mean arterial pressure and glomerular filtration rate in SLE with APS were significantly higher than those in SLE without APS (all P＜0.05). In 8 out of 11 cases (72.7%), APS nephropathy (APSN) in kidney biopsy was found, characterized by small vessel vaso-occlusive lesions. These included thrombotic microangiopathy (TMA), fibrous intimal hyperplasia (FIH), focal cortical atrophy (FCA) and tubular thyroidization. Among those, 5 cases (45.5%) had chronic APSN and 4 (36.4%) had acute APSN (one case had acute and chronic APSN at the same time). The incidences of APSN and acute APSN in the SLE with APS group were significantly higher than those in SLE without APS group (P＜0.05). Conclusions The major renal manifestations of SLE with APS are hypertension, nephrotic level of proteinuria and renal insufficiency. Other than lupus nephritis, also a high incidence of APSN is found in SLE with APS patients.

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Efficacy of low-dose daytime ambulatory peritoneal dialysis in diabetic end-stage renal disease patients with better residual renal function

Jun JI ; Xiaoqiang DING ; Yi FANG ; Yisheng SHAN ; Jie TENG ; Chensheng FU ; Min YUAN ; Liming CHEN

Chinese Journal of Nephrology.2010;26(11):824-828. doi:10.3760/cma.j.issn.1001-7097.2010.11.005

Objective To study the efficacy of low-dose daytime ambulatory peritoneal dialysis (DAPD) and low-dose CAPD in diabetic end-stage renal disease (ESRD) patients with better residual renal function (RRF). Methods Forty stable diabetic ESRD patients with better RRF (rGFR ≥ 5 ml/min and urine volume ≥ 750 ml/d) were enrolled. They were randomly divided into two groups: low-dose DAPD group (n=20) and low-dose CAPD group (n=20). DAPD group received three 1.5 L to 2 L daily exchanges with a nocturnal empty belly, dwelling for 3 to 4 hours. CAPD group received three 1.5 L to 2 L daily exchange or four 1.5 L daily exchange regimens and dwelled during the night. At the beginning of the study and 6 months later, total weekly Kt/V and Ccr (peritoneal+renal), rGFR were calculated. Meanwhile 24-hour urinary protein,serum albumin (Alb), hemoglobin (Hb), fasting plasma glucose, glycosylated hemoglobin and insulin dosage were measured. Nutritional status was assessed by SGA. Results Thirty-five patients fulfilled the study. There were no significant differences between two groups in age, gender, BMI,PD time, D/Pcr, etc. At the end of the 6th month, the insulin dose[(33.6±10.9) U/d] and 24-hour dialysate protein [(11.13t4.95) g] in CAPD group were significantly higher as compared to DAPD group [(20.6±6.2) U/d, P＜0.05 and (5.66±2.88) g, P＜0.01 respectively]. Alb in CAPD group [(29.7±4.2) g/L] was significantly lower than that in DAPD group [(36.5 ±3.9) g/L, P＜0.05].While the net ultrafiltration [(554±187) ml vs (309±177) ml], 24-hour urine volume [(1090±361)ml vs (750±258) ml] and rGFR [(8.21±2.40) ml/min vs (4.88±2.11) ml/min] in DAPD group were all significantly higher than those in CAPD group (all P＜0.05). Conclusion For the diabetic ESRD patients with better RRF, the low-dose DAPD regimen is more effective to control plasma glucose, improve nutritional status and protect RRF than the low-dose CAPD.

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Association of complement C1q gene polymorphism with the susceptibility to lupus nephritis in the people of Wuhan district, Hubei province, China

Ling FU ; Jianhua PAN

Chinese Journal of Nephrology.2009;25(12):912-915. doi:10.3760/cma.j.issn.1001-7097.2009.12.007

Objective To investigate the association of complement C1q polymorphism with the susceptibility to lupus nephritis (LN) in the people of Wuhan district,Hubei province, China. Methods The polymorphie frequency at C1q region of accommodation Aexon2, Bexonl,Bexon2,Cexon2 in 30 LN patients,20 non-LN nephropathy controls and 10 healthy controls were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)and then the sequence of mutational site was detected directly.Association of C1q polymorphism with LN was analyzed by chi-square criterion. Results Already repoaed mutable points oversea,such as C1qAexon2:C→T,C1qBexon1:G→A,C1qBexon2:G→A,C1q Cexon2:C deletion,in C1q region of accommodation were not detected in 30 LN patients,20 nonLN nephropathy controls and 10 healthy controls. Conclusion Polymorphism in the regulatory region of C1q region of accommodation is not associated with the susceptibility to LN in the people of Wuhan district,Hubei province,China,which may be influenced by a small number of subjects.

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Association of pre-transplant serum level of anti-endothelial cell antibody with acute rejection in kidney transplant recipients

Fei HAN ; Rong LV ; Juan JIN ; Jianyong WU ; Ying CHEN ; Huiping WANG ; Jianghua CHEN

Chinese Journal of Nephrology.2009;25(12):896-900. doi:10.3760/cma.j.issn.1001-7097.2009.12.003

Objective To study the pre-transplant serum level of anti-endothelial cell antibody(AECA)in kidney allograft recipients and its impact on the episode of acute rejection (AR) within 6 months after transplantation. Methods A total of 495 kidney allograft recipients with pre-transplant serum between December 1998 and August 2003 in our center and 40 healthy controls(negative controls)were enrolled in the study.Clinical data including AR within 6 months after transplantation were analyzed retrospectively.The serum AECA level was measured by cyto-ELISA using EA.hy926 cells as substrate,which was shown as the ratio of P (patient)/N (negative control)=(A_(petient)-A_(blank contrnal)/(A_(negative contral)-A_(blank contral).AECA was considered positive when P/N value Was greater than the average A_(negative control)value plus two times the standard deviation.Results Positive rate of AECA was 18.8%(93/495).AECA level in hemodialysis patients who had been on hemodialysis more than 12 months was 1.43±0.37,greater than those less than 12 months(1.27± 0.32,P=0.013)and those of non-dialyzed patients(1.31±0.32,P=0.029).Correlation coeffieient between AECA level and hemodialysis duration was 0.218 (P=0.018).AR incidence in AECA positive recipients was 38.7%,greater than that in AECA negative recipients (23.4%,P=0.002). Incidence of acute T cell-mediated rejection and acute antibody-mediated rejction increased significantly (P=0.035,P=0.002 respectively).Multifactor logistic regression analysis indicated that AECA positive,PRA greater than 1 0%and high CDC level were risk factors of AR with odds ratio of 2.056,1.751 and 1.764 respectively(P=0.004,0.029,0.050). Conclusions The AECA positive in pre-transplant serum indicates the elevated risk of acute allograft rejection.The AECA level increases with prolonged hemodialysis duration.

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Effect of 1,25(OH)_2D_3 on the expression of vitamin D receptor, tumor necrosis factor a and transforming growth factor β1 in rat peritoneal mesothelial cells stimulated by lipopolysaccharide

Lina YANG ; Jianfei MA ; Shuo CHEN ; Liyan LI ; Lining WANG

Chinese Journal of Nephrology.2009;25(11):856-861. doi:10.3760/cma.j.issn.1001-7097.2009.11.010

Objective To observe the effect of 1,25(OH)_2D_3 on expression of vitamin D receptor (VDR), tumor necrosis factor α (TNF-α) and transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells (RPMCs) stimulated by lipopolysaccharide (LPS), so as to provide some evidence for clinical use of 1,25 (OH)_2D_3 on peritoneal dialysis-associated peritoneal inflammation. Methods RPMCs were isolated, cultured and passaged by enzymaticdisaggregation, then identified by phase contrast inverted microscope, transmission electron microscope with immunocytochemistry method. RPMCs were incubated with LPS (1,10,100 mg/L) and LPS (10 mg/L) for 2,6,12 hours, or stimulated by 1,25(OH)_2D_3 (10~(-8) mol/L, 10~(-7) mol/L, 10~(-6) mol/L) after incubated with LPS (10 mg/L) for 2 hours. RPMCs in the control group were justincubated with medium. Expression of VDR mRNA and protein was detected by RT-PCR and Western blot. In addition, ELISA was performed to investigate the changes of TNF-α and TGF-β1 in culture medium. Results Compared with control group, the expression of VDR mRNA and protein was decreased in LPS group (P<0.05). LPS could significantly induce the expression of TNF-a and TGF-β1 in RPMC (P <0.01), which could be partially reversed by different concentrations of 1,25 (OH)_2D_3 (P<0.01). Conclusions 1,25 (OH)_2D_3 can reverse the decrease of VDR mRNA and protein induced by LPS as well as the induction of TNF-a and TGF-β1 up-regulated by LPS in RPMC in a dose- and time-dependent manner. Our research provides experimental evidence of VDR ameliorating peritoneal dialysis-associated peritoneal inflammation and peritoneal fibrosis.

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Correlation between the ratio of intima/media thickness and inflammatory factors in renal small artery of diabetic nephropathy rats

Anju ZHAO ; Songmin HUANG ; Santao OU ; Zhejun CHEN ; Xueli LAI ; Fang LIU ; Wanxin TANG

Chinese Journal of Nephrology.2010;26(10):775-779. doi:10.3760/cma.j.issn.1001-7097.2010.10.011

Objective To observe the change of intima/media thickness ratio and expression of inflammatory factors in renal small artery of diabetic rats, and to explore the correlations of intim/media ratio with inflammatory factors and vascular lesions of diabetic nephropathy (DN) rats. Methods Seventy healthy SD rats were randomly divided into diabetic nephropathy group (DN, n=40) and normal control group (N, n=30). DN rat model was induced by intraperitoneal injection of streptozotocin (STZ). Thirty-five DN rats were successfully established. N group received same dose of citrate buffer. Rats were sacrificed after 4, 12, 24 weeks respectively.The intima/media thickness ratio in renal small artery was detected by immunofluorescence. The monocyte chemoattractant protein-1 (MCP-1) protein and mRNA expression of renal small artery were detected by immunohistochemistry and in-situ hybridization at each time point. Results Blood glucose and urine protein excretion (24 h) at different time points in DN group were significantly higher than those of N group (P＜0.05). From the 12th week, Scr, BUN, serum phosphorus were significantly higher than those of N group (P＜0.05). At the 4th week, renal small artery had the expression of MCP-1 protein and mRNA. The expression increased gradually with time, reached the highest at the 24th week, and was significantly higher than that of N group at each time point (P＜0.05). Immunofluorescence results showed that as compared to N group, in the first 4 weeks, intima/media thickness ratio in DN group was not different, at the 12th week the ratio was higher but without significant difference, at the 24th week the ratio was significantly higher (P＜0.05). Small artery intima/media thickness ratio of DN group was positively correlated with MCP-1, cholesterol, triglyceride, serum phosphate (r=0.742, P＜0.01; r=0.740, P＜0.01; r=0.829, P＜0.01; r=0.580, P＜0.01). Conclusions The arterioles intima/media thickness ratio of early DN is significantly correlated with MCP-1, lipids and phosphorus. MCP-1 may be involved in the DN vascular disease.

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