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Effect of bone marrow stem cells mobilization by SCF combined with G-CSF on renal regeneration and repair in UUO rats

Jianjiang ZHANG ; Zhuwen YI ; Xiaojie HE ; Qingnan HE ; Xiqiang DANG ; Danlin HUANG ; Yan CAO ; Xiaochuan WU ; Shuanghong MO

Chinese Journal of Nephrology.2009;25(9):711-717. doi:10.3760/cma.j.issn.1001-7097.2009.09.013

Objective To investigate the effect and possible mechanism of bone marrow stem cell mobilized by stem cell factor (SCF) with granulocyte colony-stimulating factor(G-CSF)on renal peritubular capillary, fibrosis and renal function in unilateral ureteral obstruction (UUO) rats. Methods One hundred and twenty eight healthy male Wistar rats were randomly divided into four groups: Sham group, SCF-G group, UUO group and UUO+SCF-G group. Eight rats of each group were randomly selected and killed on the 5th, 14th, 21st and 28th day. Serum creatinine, CD34 positive cells and factor Ⅷ positive cells in renal interstitium, histopathologic lesion scores of interstitial fibrosis and interstitial pathology in kidney were measured. The mRNA expression of vascular endothelial growth factor (VEGF). and thrombospondin-1 (TSP-1) in the renal cortex was detected. Results (1) The renal interstitial fibrosis anti the loss of peritubular capillary were observed in UUO group after two weeks. (2) The number of bone marrow stem cells homing to renal interstitium in UUO +SCF-G group was significantly higher than that in UUO and Sham groups (P<0.05). (3) The loss of peritubular capillary in UUO+SCF-G group appeared later than that in UUO group (P<0.05). (4) The interstitial fibrosis and tubule injury was milder in UUO+SCF-G group than that in UUO group (P<0.05). (5) The decrease of VEGF mRNA expression of renal cortex in UUO +SCF-G group was seen later than that in UUO group. VEGF mRNA expression in UUO+SCF-G group was higher than that in UUO group. (6) The increase of TSP-1 mRNA expression of renal cortex in UUO+SCF-G group was seen later than that in UUO group. TSP-1 mRNA expression in UUO+SCF-G group was lower than that in UUO group (P<0.05). (7) In UUO and UUO+SCF-G groups, peritubular capillary index was negatively correlated with serum creatinine, interstitial fibrosis and interstitial lesion scores. VEGF mRNA expression of renal cortex was positively correlated with peritubular capillary index, and TSP-1 mRNA expression of renal cortex was positively correlated with peritubular capillary index. Conclusions (1)The loss of peritubular capillary is found in UUO group, and is correlated with interstitial fibrosis and interstitial lesion. (2) Application of SCF with G-CSF can effectively motivate stem cells to injured renal tissue, contribute to decrease the loss of peritubular capillary, lessen interstitial fibrosis and interstitial lesion, and ameliorate renal function. (3) Application of SCF with G-CSF can up-regulate VEGF mRNA expression and down-regulate TSP-1 mRNA expression, which may contribute to promote the repair of endothelial cells and protect peritubular capillary.

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Association between non-dipping circadian blood pressure rhythm and left ventricular hypertrophy in chronic kidney disease patients

Xiajing CHE ; Zhaohui NI ; Weiming ZHANG ; Minfang ZHANG ; Leyi GU ; Yucheng YAN ; Hua YING ; Chunhua HU ; Jiaqi QIAN

Chinese Journal of Nephrology.2009;25(9):663-667. doi:10.3760/cma.j.issn.1001-7097.2009.09.001

Objective To clarify the association between non-dipping circadian blood pressure (BP) rhythm and left ventrieular hypertrophy (LVH) in chronic kidney disease (CKD) patients. Methods A total of 257 CKD patients of stage 1 to 5 were enrolled in the study. The parameters of BP and circadian rhythm were measured by ambulatory BP monitoring (ABPM) and the cardiac structure was examined by echocardiography. The association between circadian BP rhythm and echocardiographic parameters was studied. Results The prevalence of abnormal circadian BP rhythm (non-dipping rhythm) was quite high (75.4%) in CKD patients and increased with the deterioration of renal function. Even if in the normal BP group, the prevalence of non-dipping rhythm was 71.3%. The change of cardiac structure such as LVH in non-dipping patients was more obvious than the dipping patients. The left ventrieular mass index (LVMI) was positively correlated with BP, non-dipping rhythm. Multiple regression analysis showed that 24 h-SBP (β=0.417, P<0.01), triglyceride (TG) (β=-0.132, P=O.007), Hb (β=-0.394, P=0.016) and gender(β=0.158, P=0.039) were independent risk factors of LVMI. Conclusions The prevalence of non-dipping rhythm is quite high in CKD patients and increases with the deterioration of renal function. The change of cardiac structure such as LVH is obvious in CKD patients, especially in non-dipping group. The non-dipping rhythm is related with LVMI.

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Impact of pentoxifylline on the pathway of high glucose-induced expression of connective tissue growth factor in mesangial cells

Jingyun ZHANG ; Demin YU ; Chunjun LI ; Mingzhen LI ; Qiumei ZHANG ; Pei YU

Chinese Journal of Nephrology.2009;25(8):640-645. doi:10.3760/cma.j.issn.1001-7097.2009.08.011

Objective To investigate the effect of pentoxifyccine (FIX) on the pathway of high glucosd-induced expression of CTGF in mesangial cells. Methods Cultured rat mesangial celld were used to study the influence of different concentration of high glucose on the expression of TGF-β, CTGF, p-Smad2/3, Smad7 and FN in different exposure time. Furthermore the effect of high glucose plus TGF-β neutral antibody and different concentration of FIX on the obove expression was evaluated as well. Results High glucose could increase TGF-β, CTGF mRNA and protein expression in mesangial cells (P<0.05) in time-and dose-dependent manner, and at the same time p-Smad2/3 expression increased and Smad7 expression decreased (P<0.05).The blockage of TGF-β could decrease high glucose-induced CTGF mRNA and protein expression by 86.4% and 91.8%. PTX could suppress high glucose-induced CTGF expression in mesangial cells. When the PTX dosage increased, the suppressive effect became more remarkable, but PTX had no influence on the TGF-β expression. Conclusions High glucose up-regulates CTGF mRNA and protein expression mainly through TGF-β-Smads pathway. PTX can suppress CTGF expression effectively, but has no direct inhibition of TGF-β expression.

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Effect of high glucose on the expression of liver X receptors and ABCA1 in human THP-1 macrophages

Yixin QIAN ; Zhaohui NI ; Leyi GU ; Huili DAI ; Yucheng YAN ; Weiming ZHANG ; Ling WANG ; Zanzhe YU ; Qingwei WU ; Jiaqi QIAN

Chinese Journal of Nephrology.2009;25(8):630-634. doi:10.3760/cma.j.issn.1001-7097.2009.08.009

Objective To investigate the effect of high glucose on the expression of liver X receptors (LXRs) and ATP-binding cassette transporter A1 (ABCA1) in human macrophages (THP-1 cell line). Methods THP-1 monocytes were differentiated into macrophages by induction of phorbol 12, 13-dibutyrate (PMA). Surface markers of macrophages were identified by CD68 immunohistochemistry. The macrophages were cultured with different concentration (5.6, 11.1, 22.2 and 33.3 mmol/L) of glucose and different time (0, 0.5, 2, 6, 12, 24, 48, 72 h). Real time PCR and Western blotting methods were used to examine the mRNA and protein expression of LXRs and ABCA1. Results As compared to 5.6 mmol/L glucose, macrophage LXRβ and ABCA1 were decreased significantly at both mRNA and protein levels in dose-and time-dependent manner (P<0.05). Conclusion Hyperglycemia may play a role in the pathogenesis of arteriosclerosis through the inhibition of LXRs and ABCA1 expression in diabetic patients.

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Role of rat organic anion transporter 1 in renal cellular uptake of aristolochic acid Ⅰ and the induction of cellular toxicity

Rui ZHANG ; Xiao YANG ; Mei LIU ; Jun WU ; Yunfang ZHANG ; Wenxing PENG ; Qingyu KONG ; Xiuqing DONG ; Xueqing YU

Chinese Journal of Nephrology.2009;25(8):624-629. doi:10.3760/cma.j.issn.1001-7097.2009.08.008

Objective To investigate the role of rat organic anion transporter 1 (OAT1, SLC22A6) in the renal cellular uptake of AA Ⅰ and its impact on cellular toxicity. Methods HEK-293 cells were transfeeted with rat OAT1 cDNA or empty vectors. The over-expression of rOAT1 was confirmed by Western blot analysis and its activity was validated by using para-aminohippurate (PAH) as a probe. Cellular apoptosis was examined by flow cytometery using propodium iodode (PI) and annexin V-FITC staining. Results Concentration-and time-dependent intracellular accumulation of AA Ⅰ was observed in rOATl-transfected HEK-293 cells. After treatment with AA Ⅰ at the concentrations of 40 mg/L, 80 mg/L, 120 mg/L and 160 mg/L,respectively, for 45 min, the intracellular concentrations of AA Ⅰ in rOAT1-transfected HEK-293 cells were higher than those in controls (P<0.05). After treatment with AA Ⅰ (120 mg/L for 30 min, 60 min, 90 min and 120 min, respectively, the intracellular concentrations of AA Ⅰ in rOAT1-transfected HEK-293 cells were higher than those in controls (P<0.05). PAH significantly reduced the intracellular accumulations of AA Ⅰ in rOAT1-transfected HEK-293 cells. After treatment with AA Ⅰ at the concentrations of 40 mg/L, 80 mg/L, 120 mg/L and 160 mg/L respectively for 35 min, the intracellular accumulations of AA Ⅰ in rOAT1-transfected HEK-293 cells that treated with PAH were lower than those that were not treated by PAH. Cellular apoptosis and caspase-3 expression in rOAT1-transfeeted HEK-293 cells were significantly up-regnlated as compared to controls (P<0.05). Conclusion rOAT1 is involved in the cellular uptake of AA Ⅰ which leads to increased epithelial apoptosis. Further studies are suggested to investigate the role of human OAT in the disposition of AA and its toxicological consequences.

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Correlation between serum bone metabolism biomarkers and bone mineral density in chronic kidney disease patients with different stages

Jianqing JIANG ; Shan LIN ; Zhenfeng ZHENG ; Pengcheng XU ; Junya JIA ; Jun BI

Chinese Journal of Nephrology.2009;25(8):619-623. doi:10.3760/cma.j.issn.1001-7097.2009.08.007

Objective To investigate the correlation between serum bone metabolism biomarkers and bone mineral density (BMD) in chronic kidney disease (CKD) patients with different stages. Methods Seventy-eight CKD patients were enrolled in this study and were assigned to different groups according to their ereatinine clearance (Cer). Patients with Cer ≥ 15 ml/min were divided into 4 groups based on clinical CKD 1-4 stage standard, and those with Ccr<15 ml/min were divided into two groups of hemodialysis (HD) and non-HD. Their levels of serum calcium, phosphorus, alkalinity phosphatase (ALP), urea, Ser, osteocalein (gla-protein, OC), calcitonin (CT), intact parathyroid hormone (iPTH), osteoprotegerin (OPG) and BMD were detected respectively. Results (1) The serum levels of OPG, iPTH and phosphorus increased significantly in stage 3, 4, 5, respectively (P<0.01), and serum OPG level was elevated to (5.1±1.34) ng/L after HD, which was significantly higher than (3.35±0.76) ng/L before HD (P<0.05). The levels of serum OC, CT, calcium, ALP were not significantly different among patients with different CKD stages, while the level of OC was elevated in HD patients (P<0.05). The femoral WARDS triangle BMD of CKD stage 4 patients decreased to 0.77±0.09, which was less than the value of CKD stage 1 patients (P<0.01), with litde influence from hemodialysis treatment. (2) The level of serum OPG was positively correlated with the levels of serum phosphorus, iPTH, OC (r = 0.51, 0.39, 0.36,all P<0.01), and it was negatively correlated with the level of Ccr (r =-0.70, P<0.01). The femoral WARDS triangle BMD was negatively correlated with the levels of iPTH, OC, phosphorus and OPG (r =-0.59,-0.51,-0.45,-0.48, all P<0.05). Conclusions Most of serum bone metabolism biomarkers change according to the declined level of Cer. Compared with serum phosphorus, the levels of iPTH, BGP and femoral WARDS triangle BMD, serum OPG may be early diagnosticmarkers of renal osteodystrophy in CKD patients.

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Dexamethasone inhibits puromycin aminonucleoside-induced injury of mouse glomerular podocytes via stabilizing the expression and distribution of podocin in vitro

Yueqiang WEN ; Li YU ; Jie WEN ; Zhihong HAO ; Rongyan CHEN ; Lina WANG

Chinese Journal of Nephrology.2009;25(7):509-513. doi:10.3760/cma.j.issn.1001-7097.2009.07.004

Objective To observe the effects of puromycin aminonucleoside (PAN) and dexamethasone (DEX) on the expression and distribution of pedocin in vitro, and to explore the possible mechanism of DEX in improving proteinuria. Methods Mouse podecyte cells (MPCs) in control group were cultured with RPMI-1640 plus 0.02% DMSO, and were subjected to PAN treatment alone (PAN group) or PAN plus DEX (DEX group) for 8, 24,48 hours respectively. The pedocyte morphology was observed by phase-contrast microscope, and was analyzed by Image J. The distribution, mRNA and protein expression of podocin were detected by indirect immunocytofluorescence, semi-quantitative RT-PCR and Western blot, respectively. Results The well-developed arborization and interconnection of podocytes were found in control group. PAN treatment led to significant shrinkage of pedocytes with decreased distribution at 43% of control group at 8 h, 10% at 24 h and 5.7% at 48 h (P<0.01), respectively, together with podocyte foot process retraction as well as effacement and loss of cell contact. RT-PCR revealed podoein mRNA expression prone to decrease. Western blot showed podoein protein expression was significantly decreased and immunocytochemistry revealed podoein expression was disappeared in the cellular membrane after PAN treatment. DEX significantly prevented the shrinkage of podcytes, with decreased area at 43.9% of control at 8 h, 26.2% at 24 h and 29.6% at 48 h (P<0.05), respectively, and up-regulated the mRNA and protein expression of podocin at 48 h (P<0.05). The abnormal distribution of podocin was also alleviated by DEX. Conclusion DEX exerts a direct action on podocyte via stabilizing mRNA, protein expression and distribution of podocin, which may be associated with the improvement of proteinuria.

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Aldosterone modulates the production of matrix metalloproteinase-2 and -9 via transforming growth factor β1 signaling pathway in podocytes

Zhan FANG ; Xifeng SUN ; Fangfang HE ; Zhonghua ZHU ; Chun ZHANG

Chinese Journal of Nephrology.2009;25(7):503-508. doi:10.3760/cma.j.issn.1001-7097.2009,07.003

objective To assess the effect of aldosterone on the production of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9)and collagen Ⅳ in culture supematants of podocytes and the possible molecular mechanisms involved in the influence of aldosterone on the synthesis and degradation of extracellular matrix produced by podocytes. Methods Podecytes were treated with aldosterone at the concentration of 10-11, 10-9, 10-7 mol/L respectively. Cultured podocytes were examined at 24, 48 and 72 hours respectively. Spironolactone, a receptor antagonist of aldosterone, was added to observe the blocking effect on aldosterone. An inhibitor of TGF-β1 receptor was used to determine whether the effect of aldosterone on podocytes were mediated through TGF-β1 system. The enzymatic activities of MMP-2 and MMP-9 were assayed by gehtin zymography. Collagen Ⅳ 0.5 chain and TGF-β1 proteins released into culture supematants were assessed by Western blot and ELISA analysis. The adhesion rate of podocytes was monitored by flow cytometry. Results Aldosterone increased the activities of MMP-2 and MMP-9 in a dose- and time-dependent manner (P<0.05). Aldosterone decreased the level of collagen Ⅳ or5 chain protein in culture supernatants (P<0.05). Meanwhile, the expression of TGF-β1 was also increased (P<0.05). Spironolactone completely abolished the above-mentioned changes(P< 0.05). Blockage of TGF-β1 signaling with SB431542 prevented the aldosterone-induced upregulation of MMP-2 and MMP-9 as well as the downregulation of the collagen Ⅳ α5 chain protein and the adhesion rate of podocytes (P<0.05). Conclusions Aldosterone increases the activities of MMP-2 and MMP-9 but decreases the expression of collagen Ⅳ α5 chain and the adhension rate of podocytes possibly via TGF-β1 signaling pathway. Such alterations may contribute to glomerular podocyte injury associated with the GBM abnormality caused by the imbalance between matrix synthesis and degradation.

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Association of peritoneal angiogenesis with angiopoietin-2 in peritoneal dialysis rats

Jiangzi YUAN ; Wei FANG ; Zhaohui NI ; Aiwu LIN ; Xinghui LIN ; Jiaqi OIAN

Chinese Journal of Nephrology.2009;25(6):415-419. doi:10.3760/cma.j.issn.1001-7097.2009.06.004

Objective To investigate the association between angiopoietin-2 (Angpt-2) and peritoneal angiogenesis in a uremic peritoneal dialysis (PD) rat model. Methods Uremic (subtotal nephrectomy) rats were established and divided into non-PD, 10 d-PD, 28 d-PD and 56 d-PD groups. Standard PD solution was applied in the study. Rats undergone sham operation without PD were used as control group. Vessel density of the peritoneum was detected and quantified with anti-CD31 immunohistochemical staining. Expressive levels of Angpt-2 and vascular endothelial growth factor (VEGF) were examined in the peritoneum by real-time PCR and Western blotting. Results The non-PD group was characterized by increased vessel density in the peritoneum compared with that of the control group [(5±3)/HP vs (1±1)/HP]. Progressive angiogenesis was found in 10 d-PD, 28 d-PD and 56 d-PD groups [(10±5)/HP, (17±5)/HP, (19±4)/HP]. Furthermore, expressive levels of Angpt-2 and VEGF increased significantly in the non-PD group compared with the control (P<0.01), and such expressions were significantly higher in the PD groups as compared to non-PD group (P<0.01), but no difference was found among the PD groups. Both VEGF and Angpt-2 levels were positively correlated with vessel density(r=0.7756, P<0.01; r=0.5223, P<0.05). Conclusions Uremia and PD promote peritoneal angiogenesis in rats. Increased expression level of Angpt-2 in peritoneum is positively correlated with peritoneal angiogenesis. Angpt-2 may be a new therapeutic target of peritoneal angiogenesis.

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SHARP-2 gene silence by lentiviral-based short hairpin RNA interference prolongs survival time of rat kidney transplant recipients

Zhangfei SHOU ; Huaying XIAO ; Yifang XU ; Yuanzhi WANG ; Yi YANG ; Hong JIANG ; Huiping WANG ; Ying CHEN ; Yiwen LI ; Jianghua CHEN

Chinese Journal of Nephrology.2009;25(6):458-464. doi:10.3760/cma.j.issn.1001-7097.2009.06.015

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.

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