MicroRNA-503 regulates high-glucose induced apoptosis of renal tubular epithelial cells by targeting Bcl-2

Objective To investigate the expression vibration of microRNA-503(miR-503) and its effect on target gene Bcl-2, caspase enzyme activity and apoptosis of human renal tubular epithelial cells (HK-2) induced by high glucose, and to clarify the pathogenesis of renal tubular injury induced by high glucose. Methods HK-2 cells were cultured in normal glucose group (NG), mannitol hypertonic control group (MA), and high glucose group (HG). The morphology of apoptotic cells was observed using inverted microscope. The expression of miR-503 was determined using real-time quantitative PCR. The apoptosis rate of HK-2 cells was detected by Annexin Ⅴ-FITC double dye using flow cytometry instrument. The expression of Bcl-2 and cleaved caspase-9 were detected by Western blotting. Results In the high glucose and mannitol groups HK-2 cell, an obviously increased apoptotic rate was observed under inverted microscope compared with normal glucose group (P＜0.05). MA and HG up-regulated miR-503 expression (P＜0.01), down-regulated anti-apoptotic protein Bcl-2 expression (P＜0.05) and up-regulated cleaved caspase-9 (P＜0.05). Conclusions The expression of miR-503 increases in HK-2 cells cultured by high glucose and mannitol. MiR-503 promotes apoptosis of HK-2 cells via activating mitochondrial apoptotic pathways and enhancing cleaved caspase-9 for Bcl-2 insufficiency. The tubular toxicity of high glucose is partly due to osmotic pressure. The miR-503 may be involved in diabetic tubular injury and may be a new therapeutic target of DN.     

PPARγ配体对高糖诱导大鼠腹膜间皮细胞结缔组织生长因子和纤溶酶原激活抑制因子1表达的影响

目的 研究过氧化物酶体增殖物活化受体γ（PPARγ）天然配体15d-PGJ2及人工合成配体吡格列酮（pioglitazone）对高糖诱导大鼠腹膜间皮细胞（RPMC）表达结缔组织生长因子（CTGF）和纤溶酶原激活抑制因子1（PAI-1）的影响。 方法 胰蛋白酶消化法分离培养RPMC,经鉴定分组：（1）0.1%、1.5%、2.5%、4.25%葡萄糖作用24 h组;（2）2.5%葡萄糖作用0、6、12、24、36、48、72 h组 ;（3）0.1%、1.5%、2.5%、4.25%甘露醇作用24 h组;（4）15d-PGJ2（5、15 μmol/L）及吡格列酮（5、15 μmol/L）分别预孵育2 h,加2.5%葡萄糖再作用24 h。RT-PCR检测CTGF和PAI-1 mRNA表达;Western印迹检测PPARγ、CTGF及PAI-1 蛋白表达。 结果 正常RPMC有PPARγ表达。1.5%葡萄糖使RPMC的PPARγ蛋白表达减少（P < 0.05）,而4.25%葡萄糖作用最大（P < 0.01）;2.5%葡萄糖作用6 h,RPMC的PPARγ蛋白表达减少（P < 0.05）,而72 h达高峰（P < 0.01）。各种浓度的甘露醇作用24 h,RPMC的PPARγ蛋白表达均无明显变化（P > 0.05）。2.5%葡萄糖作用后RPMC的CTGF和PAI-1 mRNA和蛋白表达均显著增加（P < 0.01）。5 μmol/L的吡格列酮显著降低CTGF和PAI-1 mRNA和蛋白表达（均P < 0.05）,而15 μmol/L作用更强（P < 0.01）。5 μmol/L的15d-PGJ2显著降低RPMC的CTGF mRNA和蛋白表达以及PAI-1 mRNA的表达（均P < 0.05）,但不影响PAI-1蛋白表达（P > 0.05）,15 μmol/L的15d-PGJ2对CTGF和PAI-1 mRNA和蛋白表达均有抑制作用（P < 0.05或P < 0.01）。 结论 葡萄糖以时间和剂量依赖方式调节RPMC PPARγ的表达,其作用与高渗透浓度无关。PPARγ配体可显著抑制高糖诱导的CTGF和PAI-1 的表达,提示激活PPARγ可能成为防治腹膜透析相关腹膜纤维化的新途径之一。     

MiRNA-148b targeted AMPKα1 mediates high glucose-induced apoptosis in human renal tubular epithelial cells via oxidative stress

Objective To investigate the expression and mechanism of microRNA-148b (miRNA-148b) in high glucose-induced renal tubular injury. Method HK-2 cells cultured in vitro were divided into normal glucose group, mannitol hypertonic control group and high glucose group. After 48 hours of culture, the expression of miRNA-148b was detected by real-time quantitative PCR. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used for detecting production of ROS and observed under fluorescence microscope for analysis; The expression of AMPKα1, Bcl-2, NOX2, NOX4, activated caspase3 (cleaved-caspase3) were detected by Western blotting. Results Compared with the normal glucose group, the expression of miRNA-148b was up-regulated in HK-2 cells in high glucose group and hypertonic group (P＜0.01), and the production of ROS increased (P＜0.01). The expression of NOX2 and NOX4 was increased, AMPKα1 and Bcl-2 decreased, and cleaved caspase-3 was increased (all P＜0.01). Conclusions HG up-regulated miRNA-148b expression and down-regulated its target gene AMPKα1 which promotes the expression of NOX2 and NOX4 in HK-2 cells. MiRNA-148b promotes apoptosis of HK-2 cells via increasing production of ROS and enhancing cleaved-caspase3 for Bcl-2 insufficiency. The tubular toxicity of high glucose is partly due to osmotic pressure. MiRNA-148b may be involved in the pathological injury of diabetic nephropathy and is expected to become a new therapeutic target for diabetic nephropathy.     

可溶性酪氨酸激酶2融合蛋白对尿毒症腹膜透析大鼠腹膜形态和功能的影响

目的 研究可溶性酪氨酸激酶2融合蛋白（sTie-2-Fc）对尿毒症腹膜透析大鼠腹膜血管新生、溶质转运和超滤功能的影响。 方法 32只雄性Wistar大鼠按数字随机法分为假手术组、尿毒症组、尿毒症腹透组和sTie-2-Fc干预组（均n=8）。尿毒症腹透组和sTie-2-Fc干预组大鼠经腹透管每天2次腹腔灌注4.25%葡萄糖透析液（3 ml/100 g体质量）共4周,sTie-2-Fc干预组大鼠每次灌注时在透析液中加入1 μg sTie-2-Fc。各组大鼠处死前行腹膜平衡试验,检测腹膜转运和超滤功能,取大网膜标本行抗CD31免疫组化染色并计血管数。 结果 与假手术组大鼠相比,尿毒症组大鼠的2 h腹透液和血肌酐比值（D/Pcr）增高（0.78±0.05比0.70±0.09,P = 0.028）,腹透液2 h与0 h葡萄糖比值（D/D0）降低（0.69±0.05比0.76±0.07,P = 0.033）,腹膜超滤量（UF,ml）减少（2.29±0.50比4.58±1.64,P = 0.005）,腹膜血管数量增加[（5.8±3.0）/HP比（1.6±0.5）/HP,P < 0.01]。尿毒症腹透组大鼠的溶质转运较尿毒症组大鼠进一步增高（D/Pcr: 0.89±0.05比0.78±0.05,P < 0.01;D/D0:0.47±0.09 比0.69±0.05, P < 0.01）,UF（ml）减少（0.40±0.59比2.29±0.50,P = 0.005）,腹膜血管数量增多[（16.7±1.2）/HP比（5.8±3.0）/HP,P < 0.01]。干预组大鼠使用sTie-2-Fc后,UF（ml）较尿毒症腹透组大鼠显著增加（1.56±0.48比0.40±0.59,P = 0.014）,腹膜血管数量显著减少[（9.2±1.2）/HP比（16.7±1.2）/HP,P ＜ 0.01],但两组大鼠的D/Pcr和D/D0差异均无统计学意义。 结论 sTie-2-Fc使尿毒症腹透大鼠腹膜血管新生减少,超滤增加,有利于保护腹膜结构和功能,可能是防治腹透后腹膜结构和功能改变的另一靶点。     

细胞因子信号传导抑制蛋白1抑制高糖诱导的肾小球系膜细胞单核细胞趋化蛋白1的表达

目的 探讨细胞因子信号传导抑制蛋白1(SOCS-1)对高糖状态下肾小球系膜细胞单核细胞趋化蛋白1（MCP-1）表达的影响。 方法 体外培养人肾小球系膜细胞,应用脂质体2000分别转染pCR3.1-SOCS-1表达质粒和pCR3.1 空质粒载体,G418筛选阳性克隆。分别采用低糖（5.5 mmol/L）、高糖（30 mmol/L）、低糖+甘露醇（24.5 mmol/L甘露醇）和JAK-STAT信号通路抑制剂AG490 （10 μmol/L）进行刺激。Western印迹检测系膜细胞SOCS-1、信号转导和转录活化因子1、3（STAT1、STAT3）及其磷酸化蛋白（p-STAT1、p-STAT3）的表达。ELISA法和放免法测定细胞上清液中MCP-1、FN和Ⅳ型胶原的含量。RT-PCR法检测SOCS-1和MCP-1 mRNA的表达。 结果 高糖刺激系膜细胞SOCS-1蛋白和mRNA表达呈时间依赖性变化, 4 h表达达到峰值,然后逐渐减低,24 h达基线水平。与低糖组相比,高糖组系膜细胞STAT1和STAT3磷酸化水平显著上调（P < 0.01）; MCP-1 mRNA水平表达显著上调[（0.39±0.05）比（0.16±0.02）,P < 0.01];上清液中MCP-1[（459±67）比（241±19） ng/L]、FN[（5.84±0.61）比（3.41±0.31） mg/L]和Ⅳ型胶原[（16.45±2.30）比（9.56±1.52） μg/L] 含量均显著增加（均P < 0.01）。与空载体对照组相比,SOCS-1过表达组系膜细胞STAT1和STAT3的磷酸化水平显著下降（P < 0.05）;MCP-1 mRNA表达下调[（0.34±0.04）比（0.42±0.05）,P < 0.05]; 上清液中MCP-1[(387±47）比（463±56） ng/L]、 FN[（4.61±0.57）比（5.76±0.74） mg/L]和Ⅳ型胶原[(13.4±2.32)比（17.1±2.57） μg/L] 含量显著减少（均P < 0.05）。与高糖组相比,AG490组系膜细胞MCP-1 mRNA（0.31±0.04）表达显著下调;上清液中MCP-1[（361±53） ng/L]、FN[（5.46±0.71）mg/L]和Ⅳ型胶原[（15.2±1.97） μg/L]含量均减少。 结论 SOCS-1过表达抑制高糖状态下肾小球系膜细胞MCP-1及细胞外基质的分泌可能部分是通过影响STAT1和STAT3的激活而实现。     

Effects of cyclooxygenase - 2 inhibitor on peritoneal function and angiogenesis in uremic peritoneal dialysis rats

Objective To investigate the effects of the cyclooxygenase-2 (COX-2) inhibitor (celecoxib) on angiogenesis and peritoneal function of uremic peritoneal dialysis rats. Methods Forty - eight male SD rats were selected, and they were randomly divided into five groups: normal control group(n=8), sham operation group(n=8), uremia group(5/6 nephrectomy, n=8), PD group [4.25% PD solution, 2 weeks PD model(n=8) and 4 weeks PD model(n=8)], PD + celecoxib intervention group[treated by celecoxib(20 mg/kg) via oral gavage, n=8].The peritoneum of uremic peritoneal dialysis rats was observed in different dialysis time from peritoneal structures, functions, peritoneal tissue capillary density (microvessel density, MVD) and COX-2, vascular endothelial growth factor (VEGF) expression level, and the impacts of celecoxib on uremic peritoneal dialysis rats peritoneal angiogenesis and peritoneal function were study. Results With the conduct of the peritoneal dialysis, peritoneal thickness increased, the inflammatory cells infiltrated, peritoneal equilibration test (PET) showed that ultrafiltration volume decreased significantly (P＜0.05), the amount of glucose transport rate rised significantly (P＜0.05), but the celecoxib could improve net ultrafiltration volume (P＜0.05), and reduce the glucose transport rate (P＜0.05). The peritoneal tissue MVD and COX - 2, VEGF expression were significantly increased in uremia group and PD group compared with that in the normal control group (all P＜0.05), were significantly lower in PD + Celecoxib intervention group than that in uremia group (P＜0.05). The correlation analysis showed that the level of COX-2 protein expression with MVD, VEGF protein expression was positively correlated (both P＜0.05), the level of VEGF protein expression and MVD was positively correlated (P＜0.05). Conclusions In vivo high glucose dialysate and uremia environmental can stimulate the COX-2 and VEGF expression raised, and the capillaries production increased in peritoneal tissue. Celecoxib can alleviate the change of peritoneal tissue morphology and function in long-term peritoneal dialysis rats. Celecoxib inhibits the peritoneal neovascularization of uremic peritoneal dialysis rats, possibly through inhibition of COX-2 expression to reduce the production of VEGF.     

Involvement of TGF-beta signal for peritoneal sclerosing in continuous ambulatory peritoneal dialysis

BACKGROUND: Functional failure of the peritoneal membrane is the most serious problem in long-term continuous ambulatory peritoneal dialysis (CAPD). Transforming growth factor-beta (TGF-ss) is one of the key mediators of fibrosis in some organs, and is thought to be involved in peritoneal alterations. In this study, we examined the role of TGF-beta1/TGF-ss receptors for human peritoneal mesothelial cells (HPMCs) and fibroblasts, and their interactions in CAPD patients. METHODS: HPMCs were cultured for 48 h in a medium containing normal- dose glucose (7 mM), high-dose glucose (30 mM) and mannitol as an osmotic agent, equal to 30 mM glucose. Cell proliferation was observed using the Tetra Color One assay. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). The expression of TGF-ss receptor types I and II was observed by flow cytometry. HPMCs and fibroblasts were co-cultured and assayed using transwell inserts in order to identify the effects of the high-concentration glucose solution. RESULTS: HPMC proliferation was inhibited by the high concentration of glucose but not by mannitol. The inhibition was abrogated by the neutralizing antibody for TGF-beta1. TGF-beta1 was induced by a high concentration of glucose but not by mannitol. The expression of both TGF-ss receptors was augmented in culture with the high concentration of glucose but not with mannitol. In the co-culture assay, the number of HPMCs was decreased and fibroblasts were significantly increased in culture with the high concentration of glucose. CONCLUSIONS: A high concentration of glucose induced a large amount of TGF-beta1 and enhanced the expression of TGF-ss receptors. HPMCs were sensitive to TGF-beta1 in response to a high concentration of glucose. These data suggest that TGF-beta1 from HPMCs exposed to a high concentration of glucose down-regulates the proliferation of HPMCs and accelerates peritoneal fibrosis.     

Disruption of low density lipoprotein receptor pathway is involved in peritoneal fibrosis induced by high glucose peritoneal dialysis solution

Objective To explore the potential mechanisms of low density lipoprotein receptor (LDLr) in high glucose peritoneal dialysis solution (PDS)-induced peritoneal fibrosis. Methods Human peritoneal mesothelial cells (PMCs) were applied. In pre-experiment, human PMCs were cultured with 1.5% PDS, 2.5% PDS and 4.25% PDS for 6 h, 12 h and 24 h. 4.25% mannitol was used as high osmotic pressure control. In formal experiment, PMCs were divided into the control group (treated with phosphate buffer saline) and the high glucose PDS group (treated with 4.25% PDS for 24 h). Morphological change of PMCs was observed by inverted microscope. The mRNA and protein expressions of extracellular matrix proteins such as α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (FSP-1) and collagenⅠ in PMCs were respectively measured by real-time PCR and Western blotting. The lipid accumulation was observed by oil red O staining and filipin staining, and the content of intracellular cholesterol ester was detected by high-performance liquid chromatography. The co-expression of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) with golgin was observed with immunofluorescent staining. The mRNA and protein expressions of LDLr, SREBP-2 and SCAP were respectively detected by real-time PCR and Western blotting. The mRNA and protein expressions of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E-binding protein 1 (4EBP1), and p70 S6 kinase (S6K1) were respectively detected by real-time PCR and Western blotting. Results (1) Compared with the 1.50% PDS stimulation, 4.25% PDS for 24 h intervention significantly increased the expression of LDLr in PMCs (P＜0.05), and high osmotic pressure control at 6 h, 12 h and 24 h had no statistical difference (P＞0.05). (2) Compared with those in the control group, in high glucose PDS group PMCs showed notable elongation consistent with the morphology of myofibroblasts, the expressions of α-SMA, FSP-1 and collagen Ⅰ were increased (all P＜0.05), and the intracellular cholesterol were enhanced (P＜0.05). Meanwhile, the co-expression of SCAP with golgin was enhanced, and the mRNA and protein expressions of LDLr, SREBP-2 and SCAP were up-regulated in high glucose PDS group (all P＜0.05). Further, the mRNA and protein phosphorylation of mTOR, 4EBP1 and S6K1 were increased (all P＜0.05). Conclusions The disruption of LDLr feedback regulation is involved in high glucose PDS-mediated cholesterol accumulation in PMCs by mammalian target of rapamycin complex 1 (mTORC1) pathway, which promotes the accumulation of extracellular matrix and peritoneal fibrosis.     

Changes of serum leptin levels and the influential factors in peritoneal dialysis patients

Objective To investigate the changes of serum leptin levels and the influential factors in maintenance peritoneal dialysis patients. Methods Seventy-six peritoneal dialysis patients were chosen at the time before, and 3 months, 6 months, 12 months, 18 months and 24 months after they began the peritoneal dialysis therapy, to examine body mass index (BMI), triceps skinfold thickness (TSF), abdominal circumference, homeostasis model assessment of insulin resistance (HOMA-IR), the plasma lipid profile, and leptin in the same situation. Results For 24 months, these patients showed higher serum leptin level than the values before commencing peritoneal dialysis treatment (P＜0.01). The level of leptin was positively correlated with the BMI(r＝0.412, P＜0.01), TSF(r＝0.308, P＜0.01), abdominal circumference(r＝0.284, P＜0.01), HOMA-IR(r＝0.184, P＜0.01) and TG(r＝0.288, P＜0.01), negatively corelated with the high-density lipoprotein cholesterol(HDL-C)(r＝-0.285, P＜0.01). Multiple logistic regression analysis showed that BMI (β＝0.339, P＜0.01), TG(β＝0.157, P＜0.01) and HDL (β＝-0.126, P＜0.05)were significant predictive factors for the changes of serum leptin levels. Conclusion Leptin maybe involve in the occurrence and the development of cardiovascular events like other metabolic parameters in peritoneal dialysis therapy.     

Fluorofenidone reduces renal fibrosis in diabetic kidney disease mice

Objective To investigate the effects of fluorofenidone (AKF-PD) on diabetic kidney disease in db/db mice and its possible mechanisms. Methods (1) Fifty-six mice aged 8 weeks (half male and half female), including 42 db/db mice and 14 wild-type mice were studied. Forty-two db/db mice randomly were divided into model group (mock-treated diabetic db/db mice), AKF-PD (250 mg?kg-1?d-1) treatment group and losartan (20 mg?kg-1?d-1) treatment group. Wild-type mice and model mice were treated with vehicle (0.5% sodium carboxymethylcellulose), while the treatment groups received either AKF-PD or losartan. After 18 weeks, the blood glucose and urinary albumin were measured, the pathological changes of kidney were observed by PAS staining. The protein expressions of type Ⅳ collagen and fibronectin (FN) in kidney tissue were detected by immunohistochemistry. (2) Mouse glomerular mesangial cells (MES-13 cells) were divided into six groups: normal glucose group (5.5 mmol/L glucose), hypertonic group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), AKF-PD group (25.0 mmol/L glucose+400 mg/L AKF-PD) and losartan group (25.0 mmol/L glucose+2 μmol/L losartan). After 72 h treatment, the expressions of type Ⅰ collagen, type Ⅳ collagen and transforming growth factor-β1 (TGF-β1) mRNA were detected by real-time PCR, and the content of TGF-β1 protein in the culture supernatant was detected by ELISA. Results (1) Compared with the wild type mice, model mice had increased weight, blood glucose and glomerulosclerosis index (all P＜0.01), accompanied with heavy albuminuria, glomerular hypertrophy, mesangial area expansion and deposition of collagen type Ⅳ and FN (all P＜0.01). Compared with model mice, in AKF-PD and losartan groups 24 h urinary albumin and glomerulosclerosis index decreased (all P＜0.01), glomerular hypertrophy and mesangial area expansion alleviated, and the protein expressions of collagen type Ⅳ and FN were inhibited (all P＜0.01). (2) Compared with the normal glucose group, the mRNA expressions of type Ⅰ collagen and type Ⅳ collagen increased in high glucose group, meanwhile the mRNA and protein expressions of TGF-β1 increased (all P＜0.01). In AKF-PD and losartan groups the expressions of type Ⅰ collagen, type Ⅳ collagen and TGF-β1 were inhibited as compared with high glucose group (all P＜0.05). Conclusion Fluorofenidone may play an anti-fibrotic effect in db/db mice by reducing the expression of TGF-β1 and inhibiting collagen synthesis in glomerular mesangial cells.     

The abnormal expressions of tristetraprolin and vascular endothelial growth factor family which participate in ultrafiltration failure

Objective To observe the expression of tristetraprolin (TTP) and vascular endothelial growth factor (VEGF) family, to test and verify whether lymphangiogenesis was involved in the occurrence of ultrafiltration failure (UFF) as well as angiogenesis. Methods Forty male SD rats of clean grade were selected (180-200 g). These rats were divided into five groups randomly: normal group (n=8), sham operation group (n=8), uremia group (n=8), peritoneal dialysis (PD) 2-week group (n=8), PD 4-week group (n=8). The uremic rats model was established by 5/6 nephrectomy, and of which the PD rats model was established on the basis. The rats of PD2-week group and PD4-week group were given regular PD with 4.25% peritoneal dialysis fluid in dose of 3 ml/100 g body weight. PD2-week group received peritoneal dialysis for 2 weeks, PD4-week group for 4 weeks. Before the rats were sacrificed, peritoneal equilibration test (PET) was applied to calculate the mass transfer of glucose and peritoneal ultrafiltration volume. The protein expressions of VEGF, VEGF–C in each group of rats’ parietal peritoneum were detected by immunohistochemical staining. Microvessel density (MVD) and lymphatic vessel density (LVD) of peritoneal tissue were marked and quantified with anti-CD31 antibody, anti-LYVE-1 antibody. RT-PCR was applied to detect the mRNA expressions of VEGF-A, VEGF-B, VEGF-C, VEGF-D, TTP. Western blotting was used to detect the protein expression of TTP. Results (1)PET revealed that, compared with normal group, the mass transport of glucose and the peritoneal ultrafiltration volume of both PD 2-week group and PD 4-week group elevated (P＜0.05); and compared with PD 2-week group, PD 4-week group’s elevated (P＜0.05). (2) Compared with normal group, the protein expression of CD31, LYVE-1, the count of MVD and LVD were increased in uremia group and PD4-week group (P＜0.05). Those of PD4-week groups likewise were increased compared to uremia group (P＜0.05). (3) Compared with normal group, the mRNA expressions of VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D were significantly increased in uremia group (P＜0.05); Compared with uremia group, the expressions in PD4-week group were significantly increased (P＜0.05). Compared with normal group, the mRNA and protein expressions of VEGF, VEGF-C were increased in PD 2-week group (P＜0.05); Compared with PD 2-week group, the expressions were increased in PD 4-week group (P＜0.05). (4) Compared with normal group, the expressions of TTP protein was decreased in uremia group and PD 2-week group (P＜0.05). Compared with uremia and PD2-week group, the expressions of TTP protein was significantly decreased in PD4-week group (P＜0.05). Conclusions High glucose peritoneal dialysis fluid and uremic circumstance result in the expression changes of TTP and VEGF family in a PD time-dependent manner. High glucose peritoneal dialysis liquid gives rise to angiogenesis and lymphangiogenesis, both of which lead to UFF.     

The effects and underlying mechanism of CD36 in high glucose - induced rat glomerular mesangial cells apoptosis

Objective To investigate the effects and underlying mechanism of the scavenger receptor CD36 in high glucose - induced rat glomerular mesangial cells apoptosis. Methods The mesangial cells of rats were divided into 4 groups: control group (5.6 mmol/L glucose), mannitol group (24.2 mmol/L mannitol+5.6 mmol/L glucose), high glucose group (30 mmol/L glucose), CD36 mono- antibody group (30 mmol/L glucose+CD36 mono-antibody). The intracellular ROS level was detected by confocal microscopy with fluorescent probe CM - H2DCFDA. MDA, GSH - PX, 8 - OHDGA in cell supernatant were detected. Apoptosis was determined by flow cytometry followed by Annexin V-FITC/PI double stains. The expression of CD36, Bax and Bcl-2 were detected by RT-PCR and Western blotting. Results The expression of CD36 was detected in glomerular mesangial cells. The highest level was found in high glucose group in 24 hours. There was no significant difference found between control group and mannitol group with respect to intracellular ROS generation, MDA, 8-OHDG, GSH-PX level, apoptosis rate, expression of CD36, Bax and Bcl-2 (all P＞0.05). There was no significant difference in the expression of CD36 between CD36 mono - antibody group and high glucose group (P＞0.05）. Compared to control group, the intracellular ROS generation, MDA and 8-OHDG levels, apoptosis rate, the expression of CD36 and Bax were significantly increased, the GSH-PX level and the expression of Bcl-2 were significantly lower in high glucose group (all P＜0.05). Compared to the high glucose group, the intracellular ROS generation, MDA and 8-OHDG levels, apoptosis rate, the expression of Bax were suppressed but the GSH-PX level and the expression of Bcl-2 increased in CD36 mono-antibody group (all P＜0.05). The intracellular ROS level was positively correlated with apoptosis rate, protein expression of CD36 and Bax gene, was negatively correlated with Bcl - 2 protein expression. Conclusions CD36 was involved in the high glucose induced apoptosis of mesangial cells which was potentially mediated by an increased level of oxidative stress.     

Effect of MG132 on the expression of ERK1/2 and connective tissue growth factor in rat peritoneal mesothelial cells induced by high glucose

Objective To observe the effect of MG132 on the expression of extracellular regulated kinase 1/2 (ERK1/2) and connective tissue growth factor (CTGF) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The second generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose (1.5%, 2.5%, 4.25%) for 24 hours, high glucose (2.5%) for 0, 12, 24, 48 hours,incubated with MG132 (0.5, 1, 2 μmol/L) for half an hour and then with high glucose (2.5%) for 24 hours. ERK1/2 protein was detected by Western blotting, and CTGF protein in supernatant was detected by ELISA. Results Compared with the control group, the expression of p-ERK1/2 was significantly increased in the groups stimulated by high glucose (P＜0.01), reached the peak at 24th hour (P＜0.01), and then the expression decreased at 48th hour, but still was higher than that in the normal control group (P＜0.01). CTGF protein expression of RPMCs induced by high glucose increased, in time- and dose-dependent manner (P＜0.05). MG132 could significantly decrease the expression of ERK1/2 and CTGF induced by high glucose (P＜0.05). Conclusions MG132 can decrease the expression of p-ERK1/2 and CTGF in RPMCs induced by high glucose. The ubiquitin proteasome pathway participates in the development of peritoneal fibrosis, and blocking the way may contribute to the prevention of peritoneal fibrosis.     

Effects of benazepril on the expressions of intergrin-linked kinase and α-smooth muscle actin in glomerular mesangial cells exposed to high glucose

Objective To investigate the effect of benazepril on intergrin-linked kinase (ILK) and α-smooth muscle actin (α-SMA) expression in glomerular mesangial cells induced by high-glucose. Methods The mesangial cells from SD rat (HBZY-1) were cultured conventionally and randomly divided into four groups: normal glucose (D-glucose 5.5 mmol/L, group NG), mannitol-treated group (mannitol 20 mmol/L, group MG), high glucose (D-glucose 30 mmol/L, group HG), Benazepril-treated high glucose group (D-glucose 30 mmol/L＋Benazepril 10 μmol/L, group ACEI). Cells from NG, MG, HG, ACEI gronps were harvested after 3, 6, 12, 24, 48 and 72 hours of treatment respectively. The mRNA expressions of ILK and α-SMA were detected by RT-PCR. The protein levels of ILK and α-SMA were detected by Western blotting and immunofluorescence. Results The expressions of ILK mRNA and protein in HG group were significantly increased compared with those in NG group (all P＜0.05). The increased expressions of ILK and α-SMA in HG group were time-dependent and the expression reached the peak at 48 h (ILK, P＜0.05) or 72 h (α-SMA, P＜0.01). The expressions of ILK and α-SMA in ACEI group were lower than those in HG group (all P＜0.01), but failed to rescue to the same level as those in NG. There was no significant differences of ILK expressions between MG group and NG group at the same time point (P＞0.05). The expressions of α-SMA mRNA and protein in MG were higher than that in NG (P＜0.05), which suggest that high osmotic pressure could cause the increasing of α-SMA. Conclusions Benazepril can decrease the expressions of ILK and α-SMA to inhibit the process of fibrosis in DN and mediate the phenotypic transformation of glomerular mesangial cells. The phenotypic transformation of glomerular mesangial cells in glucose may also depend on high osmotic pressure in DN.     

Role of autophagy in high glucose-induced podocyte lipid droplet accumulation

Objective To investigate the role of autophagy in high glucose-induced podocyte lipid droplet metabolism. Methods (1) Cultured, conditionally immortalized human podocytes (HPC) were divided into normal control group, high glucose group and mannitol group. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Protein level of SREBP-1 was analyzed by Western blotting. (2) HPC were cultured and divided into normal control group, high glucose group, high glucose+3-methyladenine (3-MA) group, and mannitol group. Acridine orange staining was used to observe the formation of autophagosomes. Western blotting was used to detect the protein levels of beclin-1 and LC3-II/LC3-I. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Western blotting was used to analyze the expression of SREBP-1. Results (1) Compared with the normal control group, the lipid accumulation in the high glucose group was increased and the lipid metabolism related molecule SREBP-1 was up-regulated (P＜0.05); There was no significant difference between the normal control group and the mannitol group in lipid accumulation (P＞0.05). (2) Compared with the normal group, the number of autophagosomes was increased and autophagy-related proteins beclin-1 and LC3-II/LC3-I were up-regulated in high glucose group (all P＜0.05). After intervened with 3-methyladenine, a significant decrease in autophagosomes was observed; Protein levels of autophagy-related proteins beclin-1 and LC3-II/LC3-I were decreased (all P＜0.05); The lipid droplets in the high glucose+3-MA group was decreased and lipid metabolism related molecule SREBP-1 was down-regulated (all P＜0.05). Conclusion Autophagy may be involved in the process of high-glucose-induced podocyte lipid accumulation by affecting SREBP-1 expression, and inhibition of autophagy can alleviate the high-glucose-induced podocyte lipid accumulation.     

Effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells

Objective To evaluate the effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells and to explore the possible mechanisms of KIM-1 involved in renal interstitial fibrosis of DN. Methods The rat renal tubular epithelial cells (NRK52E) were cultured in vitro and divided into five groups: Normal control group (D-glucose 5.6 mmol/L), Hypertonic group (D-glucose 5.6 mmol/L＋D-mannitol 24.4 mmol/L), High glucose group (D-glucose 30 mmol/L), Control siRNA group,KIM-1 siRNA group. ELISA assay was used to assess the levels of MCP-1 and FN in the cells supernatant; Western blotting was used to detect the protein expression of KIM-1; RT-PCR was used to detect mRNA expression of KIM-1, MCP-1 and FN. Results Compared with the control group, the protein and mRNA expression of KIM-1 in the high glucose group were increased at 12 h (P＜0.05), and reached the peak at 48 h (P＜0.05); the protein and mRNA expression of MCP-1 and FN in high glucose group were increased at 24 h significantly (P＜0.05), and peaked at 48 h (P＜0.05). Compared with the high glucose group, the protein and mRNA expressions of MCP-1 and FN in KIM-1 siRNA group were decreased (P＜0.05). Conclusions Down-regulating the expression of KIM-1 can significantly inhibit the expression of MCP-1 and FN, which suggests that KIM-1 may be involved in renal interstitial fibrosis of DN by regulating expression of MCP-1 and FN.     

High glucose promotes human glomerulus mesangial cells senescence through activating p53/p21 pathways and suppressing autophagy

Objective To observe the changes of senescence and autophagy in human glomerulus mesangial cells (HGMCs) induced by high glucose at different times, and to investigate the effects of rapamycin and 3-methyladenine (3-MA) on these changes. Methods HGMCs were cultured in vitro, exposed to high glucose (30.0 mmol/L glucose) for 12, 24, 48 and 72 h, and stimulated by high glucose with 500 nmol/L rapamycin or 2 mmol/L 3-MA for 72 h. Normal control group (5.5 mmol/L glucose) and hypertonic group (5.5 mmol/L glucose + 24.5 mmol/L mannitol) were set up. Cytomorphology changes were examined by light microscope to test whether cells were in senescent stage. The quantity of autophagosome was observed by electron microscope. The cell senescence was evaluated by β-galactosidase (SA β-gal) staining. The protein expressions of p53, p21, LC3 and p62 were determined by Western blotting. Results High glucose group gradually had larger size and more flat cytoplasm, polymorphonuclear cells and binucleate cells than control group as the stimulation times was prolonged. Compared with those in control group, SA β-gal positive cells in high glucose group after incubation for 72 h statistically were increased (P＜0.05); the protein expressions of p62, p53 and p21 in high glucose group after incubation for 48 h and 72 h were increased (all P＜0.05), while the autophagosome and the expression of LC3 decreased (P＜0.05). Compared with those in high glucose group, the expression of LC3 was increased dramatically in high glucose with rapamycin group (P＜0.05), while the protein expressions of p62, p53 and p21 decreased (all P＜0.05), and SA β-gal positive cells decreased (P＜0.05). However, there was no statistical difference between high glucose group and high glucose with 3-MA group in terms of above effects. Conclusions High glucose may induce HGMCs senescence through activating p53/p21 pathways and suppressing the activity of autophagy. Through enhanced autophagy activity with rapamycin, the expression of p53/p21 pathway was suppressed and senescence was relieved.     

Parathyroid hormone induces endothelial-to-adipocyte transition in endothelial cells by Wnt/β-catenin pathway

Objective To investigate whether elevated parathyroid hormone (PTH) levels could induce endothelial-to-mesenchymal transition (EndMT) and adipocyte transition in endothelial cells (ECs), and to determine the possible underlying mechanism. Methods (1) A rat model of secondary hyperparathyroidism and chronic kidney disease (CKD) was established. The adiposity in bone marrow was detected by oil red O staining. Immunofluorescence staining was performed to detect the expression and localization of cluster of differentiation 31 (CD31) and fibroblast-specific protein 1 (FSP1). (2) The human umbilical vein ECs were cultured in vitro. Western blotting was performed to detect protein expressions of EndMT-related markers CD31, FSP1 and α-smooth muscle actin (α-SMA) in interference groups with different PTH concentrations (0, 10-11, 10-9, 10-7 mol/L PTH for 48 h) and times (0, 12, 24, 48 h, 10-7 mol/L PTH), as well as the expression of β-catenin in interference groups with different PTH concentrations. The localizations of CD31, FSP1 and β-catenin were observed by cell immunofluorescence. Protein expressions of adipocytes markers peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α) by Western blotting and the degree of adipogenesis by oil red O staining were detected after transformed ECs were cultured in adipogenic culture medium for one week. Small interfering RNA (siRNA) was performed to silence β-catenin expression. ECs were divided into control siRNA group, β-catenin siRNA group, PTH+control siRNA group and PTH+β-catenin siRNA group. Protein expressions of CD31, FSP1 and PPAR-γ by Western blotting and the degree of adipogenesis by oil red O staining were determined. Results (1) In vivo, compared with the control, CKD rats had increased adipocytes in bone marrow (P＜0.05), and the co-expression of CD31 and FSP1 in bone marrow ECs. (2) In vitro, PTH significantly inhibited the expression of endothelial marker CD31 and increased the expressions of mesenchymal markers FSP1 and α-SMA in concentration-and time-dependent manners. These indexes in 10-7 mol/L PTH group and 0 mol/L PTH group, in 48 h group and 0 h group showed statistical differences (all P＜0.05). In PTH group ECs with 10-7 mol/L PTH for 48 h showed FSP1 accumulation in the cytoplasm and reduced expressions of CD31, and ECs had higher expressions of PPAR-γ and C/EBP-α as well as the degree of adipogenesis than those in control group (all P＜0.05). Furthermore, PTH enhanced the nuclea β-catenin protein levels in ECs in concentration-dependent. The expressions of β-catenin in 10-7 mol/L PTH group and 0 mol/L PTH group showed statistical differences (P＜0.05). β-catenin expressed in the cytoplasm in control group, while it enter into the nucleus in PTH group. Compared with those in PTH+control siRNA group, the expressions of CD31 and PPAR-γ as well as the degree of adipogenesis decreased in PTH+β-catenin siRNA group (all P＜0.05), while the expression of FSP1 increased (P＜0.05). Conclusions PTH induces ECs- to-adipocytes transition by the canonical Wnt/β-catenin signaling pathway, which might account for bone loss in CKD. Silenced β-catenin expression can inhibit PTH-induced EndMT and adipogenesis.     

Roles of AKAP1 in high-glucose induced mitochondrial fission in podocytes

Objective To investigate the roles of A kinase anchoring protein1(AKAP1)in high-glucose induced mitochondrial fission in podocytes. Methods Conditionally immortalized human podocytes were cultured in serum-free medium for 24 hours, and then exposed to different glucose concentration conditions in different time periods. The protein expressions of AKAP1 were observed by immunofluorescence, and AKAP1, dynamin related protein1 (Drp1) and phospho Ser 637-Drp1 (p-Drp1) were analyzed by Western blotting. AKAP1 siRNA was transfected to block AKAP1 expression.Podocytes were then divided into normal control group (5 mmol/L glucose), hypertonic group (30 mmol/L mannitol+5 mmol/L glucose), high glucose group (35 mmol/L glucose), and high glucose+AKAP1 siRNA group. Mitochondrial morphological changes were assessed by mitotracker red staining. Podocyte apoptosis was assessed by flow cytometry. Results Compared with normal group, high-glucose induced more podocytes apoptosis (P＜0.05), more mitochondrial fission with decreased aspect ratio and form factor (all P＜0.05). Upregulated AKAP1 protein level, and increased ratio of p-Drp1/Drp1 (all P＜0.05) in time and concentration dependent manners were also observed. Compared with high glucose group, transfection of AKAP1 siRNA showed less apoptosis (P＜0.05), less mitochondrial fission with increased aspect ratio and form factor (all P＜0.05), and down-regulated AKAP1 protein level as well as p-Drp1/Drp1 ratio (all P＜0.05). Conclusion High glucose induced mitochondrial fission might be induced through AKAP1-Drp1 pathway.     

可溶性Tie2融合蛋白对尿毒症腹膜透析大鼠血管新生的影响

目的 观察可溶性Tie2融合蛋白(sTie2/Fc)对尿毒症腹膜透析大鼠腹膜血管新生的影响.方法 48只雄性SD大鼠,按随机数字表法分为以下6组:正常对照组、假手术组、尿毒症非腹透组、4.25％腹透组、sTie2/Fc 2.5 μg/kg干预组、sTie2/Fc 5.0 μg/kg干预组.腹透组按照4.25％腹透液30...