乌索酸改善高糖诱导系膜细胞损伤的机制

目的 探讨乌索酸能否通过抑制高糖状态下系膜细胞内miRNA-21的过表达,上调其靶基因PTEN的表达,抑制磷脂酰肌醇-3-激酶(PI3K)-Akt-哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的激活,诱导自噬,减少细胞外基质堆积,发挥其肾脏保护作用.方法 高糖培养大鼠肾小球系膜细胞,以PI3K抑制剂LY294002以及不同剂量乌索酸进行干预,应用甲基噻唑基四唑(MTT)法观察细胞增殖能力,总蛋白/总细胞数测定细胞肥大,Western印迹和实时定量PCR检测PTEN-PI3K-Akt-mTOR信号通路活性、Ⅰ型胶原及自噬标志物.透射电镜观察自噬体的形成.结果 与正常对照组相比,高糖培养的系膜细胞出现显著的肥大、增殖,细胞内miRNA-21表达明显上调,PTEN蛋白及mRNA的表达明显下调,p85PI3K、磷酸化(p)-Akt、p-mTOR、Ⅰ型胶原、p62/SQSTMI表达明显增加,LC3 II表达明显降低,差异均有统计学意义(均P＜ 0.01).与高糖组相比,乌索酸干预组及LY294002组细胞肥大、增殖程度均明显降低,p85PI3K、p-Akt、p-mTOR、Ⅰ型胶原、p62/SQSTMI的表达均明显降低,LC3 II表达明显升高,差异均有统计学意义(均P＜ 0.01);但LY294002组细胞内miRNA-21和PTEN的表达与高糖组差异无统计学意义,而乌索酸干预组细胞内miRNA-21的表达明显下调,PTEN的表达明显上调,差异均有统计学意义(均P＜ 0.01).结论 乌索酸可能通过抑制高糖培养系膜细胞内miRNA-21的过表达,上调PTEN表达,抑制PI3K-Akt-mTOR信号通路异常活化,增强自噬从而减少细胞外基质堆积,减轻细胞的肥大、增殖.     

雷帕霉素通过调节mTOR-ULK1信号通路减轻高糖诱导的足细胞损伤

目的探讨雷帕霉素对高糖环境下足细胞自噬和损伤作用的机制。 方法体外培养永生化小鼠肾小球足细胞(mouse podocyte cell 5,MPC5)并进行分组：甘露醇等渗组(mannitol isotonic group,MG组)、高糖组( high glucose group,HG组)、雷帕霉素组(rapamycin group,RG组)以及自噬相关蛋白5-siRNA组(SiG组)。PCR和Western印迹检测足细胞标志Synaptopodin、自噬相关的ULK1以及mTOR通路相关蛋白p70S6K的表达。 结果与MG组相比,HG组的Synaptopodin表达降低,自噬活性降低,p-ULK1以及p70S6K表达明显升高。与HG组相比,RG组的Synaptopodin表达升高,自噬活性较高,p-ULK1以及p70S6K表达较低。SiG组表现出与HG组相似的变化趋势。 结论雷帕霉素可能通过mTOR-ULK1信号通路调节足细胞内自噬反应、减轻高糖环境引起的足细胞损伤。     

马兜铃酸损伤大鼠肾小球足细胞的研究

目的 探讨马兜铃酸是否能损害肾小球足细胞.方法 用关木通浸膏水溶液间断灌胃制作马兜铃酸肾损害大鼠模型,分别于第1、4周末检测早期马兜铃酸肾损害大鼠24 h尿蛋白量,并以十二烷基磺酸钠-聚丙烯酰胺凝胶电泳观察尿蛋白成分.随后处死大鼠取肾组织,用激光显微切割捕获技术分离肾小球;用实时荧光定量PCR检测肾小球中nephnn、podocin、CD2AP、podocalyxin、podoplanin的mRNA表达;电镜下测量肾小球足突的平均宽度.结果 模型大鼠4周末尿蛋白量较对照组显着增多(P＜0.01),其中白蛋白含量明显增加.电镜结果显示肾小球足突平均宽度较对照组显著增宽(P＜0.01).肾小球中nephrin、podocin、CD2AP、podocalyxin、podoplanin的mRNA表达均较对照组显著减少,分别下调34％、62％、56％、50％(P＜0.01)及27％(P＜0.05).结论 马兜铃酸能损伤肾小球足细胞,导致足细胞相关蛋白mRNA表达下调,足突节段增宽,并出现白蛋白尿.     

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.     

小檗碱在高糖及糖基化终末产物诱导足细胞损伤中的保护作用

目的探讨小檗碱对糖基化终末产物(AGE)和高糖诱导下足细胞损伤及其骨架蛋白的影响及机制。 方法以条件永生性人足细胞作为研究对象,于含10%胎牛血清及100 U/L γ-干扰素的RPMI 1640培养液中进行体外培养,细胞增殖并诱导分化后进行分组处理。分别用高糖(30 mmo/L)、AGE(100 μg/ml)、小檗碱(10 μmo/L)处理48 h后,激光共聚焦检测技术观察纤维状肌动蛋白(F-actin),球状肌动蛋白(G-actin)变化;原位细胞免疫组化检测cspase-3,nephrin表达。采用SPSS13.0统计软件包进行统计学分析。 结果共聚焦显微镜下观察显示,高糖及AGE作用下,足细胞F-actin出现重排,G-actin易位,小檗碱干预后有所恢复。免疫组化结果显示,对照组及高糖组几乎未见capase-3阳性表达,但高糖＋AGE组,capase-3呈阳性表达(F＝99.339,P<0.001);高糖＋AGE组nephrin表达显著降低(F＝165.84,P<0.001),与对照组及高糖组比较,差异均有统计学意义。小檗碱作用后,高糖＋AGE组capase-3的水平下降(F＝6.927,P＝0.048),nephrin表达水平升高(F＝165.84,P＝0.025),差异均有统计学意义。 结论在持续的高糖和AGE作用下,可引起足细胞骨架蛋白F-actin、G-actin重构及分布异常,并诱导足细胞凋亡,小檗碱能改善高糖和AGE引起的足细胞骨架蛋白损伤,并抑制足细胞的凋亡,其机制可能与nephrin的参与有关。     

高糖诱导足细胞发生上皮-间叶细胞转分化

目的 通过观察高糖是否引起小鼠足细胞发生上皮-间叶细胞转分化现象,探讨糖尿病肾小球损伤的可能机制。 方法 以小鼠永生化足细胞株为研究对象,予不同浓度葡萄糖（12.5、25、50 mmol/L）处理该细胞36 h,并设低糖（5.6 mmol/L）和甘露醇（50 mmol/L）处理组为对照组。采用蛋白免疫印迹和间接免疫荧光染色方法检测α平滑肌肌动蛋白 （α-SMA）、迁连蛋白（FN）、CD2相关蛋白（CD2AP）和Wilms’肿瘤1基因（WT-1）蛋白的表达。 结果 低糖（5.6 mmol/L）和甘露醇（50 mmol/L）处理条件下小鼠足细胞表达WT-1、CD2AP,基本不表达α-SMA和FN;而予不同浓度葡萄糖处理36 h后,足细胞中α-SMA和FN表达水平呈剂量依赖性上调（P < 0.05）。间接免疫荧光染色结果也显示,与低糖组相比,高糖组中 α-SMA阳性的足细胞比例显著增加（P < 0.05）。同时,蛋白免疫印迹结果还表明高糖可呈剂量依赖性方式下调WT-1和CD2AP的表达（P < 0.05）。 结论 在高糖条件下,足细胞发生向间叶细胞表型的转分化可能是引起足细胞功能失调,进而引起糖尿病肾小球损伤发生的作用机制之一。     

Rapamycin reduces podocyte adhesion damage caused by sublytic C5b-9 via autophagy activation

Objective To determine the effect of rapamycin on sublytic C5b-9-induced podocyte adhesion damage, and whether autophagy is involved in this progression. Methods Sublytic complement C5b-9 stimulation was used in vitro. Autophagosomes were viewed using electron microscopy. Western blotting was used to measure the change of autophagy-related markers. Attachment assay was used to assess the adhesion of podocyte. Confocal microscopy was used to explore the expression patterns of cytoskeletal protein F-actin. Flow cytometry was used to measure the level of adhesion-associated protein integrin α3. Results (1) For ensuring sublytic complement injury, the maximal amounts of anti-podocyte antiserum and 160×-diluted normal human serum were used without inducing cell lysis (defined as ＞5% LDH release). (2) Sublytic C5b-9 promoted autophagy in podocyte in vitro. The proautophagic effect of sublytic C5b-9 manifested in the form of accumulated autophagosomes and enhanced expression of LC3-Ⅱ. (3) Inhibition of autophagy by 3-methyadenine enhanced the effect of sublytic C5b-9-induced podocyte injury, including serious cytoskeleton damage and markedly reduced adhesion of podocyte. (4) Rapamycin treatment significantly improved the above lesions. (5) Rapamycin enhanced autophagy induced by sublytic C5b-9 in podocyte. Conclusions In summary, rapamycin can improve sublytic C5b-9-induced podocyte adhesion damage by appropriate autophagy activation. These findings provide important information for the development of appropriate protocols for the application of mTOR (mammalian target of rapamycin) inhibitors in podocytopathy.     

1,25(OH)2D3 ameliorates high glucose-induced podocyte injury via PI3K/p-Akt signalling pathway

Objective To investigate the effect of 1,25(OH)2D3 on high glucose induced podocyte injury and its signal transduction mechanism. Methods Differentiated mouse podocytes were exposed to normal glucose, high glucose, and different concentrations of 1,25(OH)2D3 or LY294002 (a selective PI3K inhibitor) for 24 h. PCR and immunofluorescent staining were used to detect nephrin, podocin, and desmin. Western blotting was used to detect protein expression of nephrin, podocin, desmin, PI3K, Akt and p-Akt. Results Compared with high glucose group, 1,25(OH)2D3 (100 nmol/L and 1000 nmol/L) significantly up-regulated the expression of podocin and nephrin in podocytes induced by high glucose (P＜0.05). Meanwhile, 1,25(OH)2D3 (100 nmol/L) significantly reduced the expression of desmin (P＜0.05). PI3K and p-Akt were obviously reduced in high glucose group. In the presence of 1,25(OH)2D3, the trends were reversed. However the above effects of 1,25(OH)2D3 were abolished when p-Akt was blocked by the PI3K inhibitor LY294002. Conclusions 1,25 (OH)2D3 can inhibit high glucose-induced podocyte injury through PI3K/p-Akt signaling pathway.     

Early autophagy activation inhibits podocytes from apoptosis induced by aldosterone

Objective To explore the protection of early autophagy activation on podocyte injury induced by aldosterone. Methods In vitro cultured mouse podocyte clones (MPC5) were treated with aldosterone for 6, 12, 24, 48 h respectively. Apoptosis of podocytes was detected by Annexin V combined with flow cytometry. After 24 h treatment with aldosterone, the existence of apoptotic body and autophagosome was observed by electron microscopy. The protein expressions of LC3, caspase?3 and nephrin were examined by Western blotting. The mRNA expression of Beclin?1 was detected by real?time PCR. Results The induction of apoptosis and autophagy by aldosterone in podocytes was in time?dependent mannner. After 24 h treatment with aldosterone, the apoptosis was increased by 26.5% (P＜0.05) and the expression of nephrin was decreased by 28.0% (P＜0.05) compared to control group. Aldosterone remarkably induced the expression of Beclin?1 at 6 h and promoted the transformation of LC3?Ⅰto LC3?Ⅱ at 12 h (P＜0.05). Compared to simple aldosterone treatment, the apoptosis rate of podocyte was increased by 39.0%（P＜0.05）and the expression of nephrin was declined by 19.5%（P＜0.05) after 3?methyladenine (3?MA) pre?treatment. Conclusions Aldosterone can induce autophagy and apoptosis in podocytes. Autophagy occurs earlier (12 h) than apoptosis (24 h). The occurrence of autophagy can inhibit the apoptosis,so the autophagy pathway may be a new research topic of glomerular disease treatment.     

高糖与高尿酸通过醛糖还原酶通路协同作用加重内皮细胞损伤

目的寻找高血糖与高尿酸血症协同加重内皮细胞损伤的共同作用靶点和分子机制,为糖尿病合并高尿酸血症患者心血管疾病的保护提供干预靶点。 方法用人脐静脉内皮细胞系(HUVEC-C)给予高糖(30 mmol/L,HG)、高尿酸(600 μmol/L,UA)和高糖(HG)＋高尿酸(UA)联合培养48 h。利用10 μmol/L阿司他丁阻断醛糖还原酶(AR)的活性。实时定量PCR检测内皮型一氧化氮合成酶(eNOS)和AR mRNA的表达;Western blot检测还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2 (NOX2)、NOX4、eNOS和AR蛋白的表达;共聚焦显微镜检测细胞内ROS的活性;一氧化氮(NO)试剂盒检测NO活性。 结果与单独HG组和UA组相比,HG＋UA共培养组明显降低eNOS mRNA水平和蛋白表达,减少NO产生,增加内皮细胞胞内ROS活性;HG＋UA共培养明显上调AR mRNA水平和蛋白表达。应用AR抑制剂能够明显增加HG＋UA组内皮细胞eNOS mRNA水平和蛋白表达,增加内皮细胞NO的分泌水平。AR抑制剂能够明显下调HG＋UA组内皮细胞NOX4的蛋白表达,对NOX2无影响;降低细胞内ROS的含量。 结论高血糖和高尿酸协同作用通过激活醛糖还原酶途径下调内皮细胞eNOS的表达,增加ROS活性,减少NO的产生,加重内皮细胞功能障碍;而抑制醛糖还原酶,能够通过抑制NOX4表达,阻断这种协同损伤作用。     

Autophagy alleviate podocytes injury induced by contrast media via oxidative stress

Objective To evaluate the effects of autophagy on oxidative stress induced by contrast media in podocytes. Methods The differentiated mouse podocytes were exposed to contrast media (Iopromide, 50 mg/L)、rapamycin (Rap, autophagy enhancer, 1 ng/L), 3-methyladenine (3-MA, autophagy inhibitor, 2 mmol/L) for 2 hours. The expression of autophagy protein LC3-Ⅱand Beclin-1 as well as oxidative stress-related proteins Catalase, MnSOD were detected by Western blot. The formations of autophagy were observed by MDC staining, and the levels of reactive oxygen species (ROS) by CM-H2DCFDA staining. Cell activity was evaluated by CCK8 assay. Results Both the levels of oxidative stress and autophagy in podocytes increased when stimulated by contrast media, the expression of LC3-Ⅱand Beclin-1 were enhanced, Catalase and MnSOD were inhibited (all P＜0.05). Rapamycin increased the expression of Catalase, MnSOD and cell activity of podocytes, reduced the generation of ROS (all P＜0.05), but in Rap group, cell activity showed no significant difference (P＞0.05). 3-MA decreased the expression of Catalase、 MnSOD and inhibited the cell activity of podocyte, increased the generation of ROS (all P＜0.05). Conclusion Autophagy protects podocyte from contrast media by the means of reducing oxidative stress.     

Global adiponectin suppress the high expression of monocyte chemotactic protein-1 induced by high glucose in NRK52E cells

Objective To investigate the effect of globular adiponectin on the high expression of monocyte chemotactic protein-1 (MCP-1) induced by high glucose in rat renal tubular epithelial cells(NRK52E), and its relationship with adiponectin receptors and p38MAPK. Methods NRK52E cells were cultured in vitro and divided into six groups: normal glucose group (NG, 5.6 mmol/L glucose), high glucose group(HG, 25 mmol/L glucose), gAd group1 (HG+gAd 2 mg/L), gAd group2 (HG+gAd 5 mg/L), gAd group3 (HG+gAd 10 mg/L), p38MAPK antagonist group：(SB, HG+SB203580 10 μmol/L). The protein expression of phosphorylated p38MAPK (p-p38MAPK), total p38MAPK (t-p38MAPK), MCP-1 and AdipoR1/AdipoR2 were examined by western blotting. The mRNA expression of MCP-1 and AdipoR1/AdipoR2 were detected by RT-PCR and real-time PCR respectively. Results Compared with NG group, the mRNA and protein expression of MCP-1 increased significantly in HG group (all P＜0.05). The phosphorylation of p38MAPK increased (P＜0.05) with no change in t-p38MAPK protein. The addition of gAd or SB203580 inhibited the unregulation of MCP-1 and p-p38MAPK induced by HG. Two kinds of adipoR,adipoR1 and adipoR2,were all detectable in NG group, and mRNA and protein expression of adipoR1 was higher than that of adipoR2 (P＜0.01). Compared with NG group, the expression of adipoR decreased in HG group, but the difference had no statistical significance(P＞0.05). Compared to HG group, the mRNA and protein expression of adipoR1 increased in gAd groups (all P＜0.01). Conclusion The gAd can dose-dependently attenuate the overexpression of MCP-1 induced by high glucose, and this protective effect may be mediated by adipoR1 and p38MAPK.     

TAK1信号通路在高糖诱导骨髓来源巨噬细胞激活中的作用

Objective To investigate the role of transforming growth factor-β activated kinase-1 (TAK1) signaling pathway in the activation of bone marrow derived macrophages (BMDM) induced by high glucose. Methods Purity of mouse BMDM was detected by flow cytometry. The mice macrophages cultured in vitro were stimulated by high glucose and treated with TAK1 specific inhibitor 5Z-7-oxozeaenol. Cells were divided into normal control group (RPMI 1640), osmolality control group (25 mmol/L mannitol), high glucose group (33 mmol/L D-glucose) and inhibitor group (33 mmol/L D-glucose+300 nmol/L 5Z-7-oxozeaenol). Immunocytochemistry and flow cytometry were used to detect macrophage subtype. The expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis Factor-α (TNF-α) mRNA were determined by real time PCR. Expressions of p-TAK1, TAK1 binding protein (TAB1), p-JNK, p-p38 MAPK and NF-κB p65 proteins were analyzed by Western blotting. Results The purity of BMDM was about 99.36%. Compared with normal control group, high glucose group had increased percentage of M1 macrophages, increased expression of MCP-1 and TNF-α mRNA (all P＜0.05). Moreover, p-TAK1, TAB1, p-JNK, p-p38 MAPK and NF-κB p65 proteins expression also increased significantly in high glucose group (all P＜0.05). After treatment with inhibitor 5Z-7-oxozeaenol, the effects induced by high glucose were inhibited (P＜0.05). Conclusions High glucose can induce M1 macrophage activation and expression of inflammatory cytokine of BMDM, which can be inhibited 5Z-7-oxozeaenol through inhibiting TAK1/MAPK and TAK1/NF-κB pathway.     

自吞噬在脂多糖诱导HL-1心肌细胞损伤中的作用

目的 评价自吞噬在脂多糖(LPS)诱导HL-1心肌细胞损伤中的作用.方法 采用随机数字表法,将培养的HL-1细胞随机分为4组(n=15):正常对照组(C组)不予任何处理,继续培养24h;LPS组在细胞培养液中加入LPS(终浓度1 μg/ml);自吞噬诱导剂纳巴霉素组(R组)在细胞培养液中加入纳巴霉素(终浓度0.2 μg/ml),孵育48 h时加入LPS(终浓度1 μg/ml);自吞噬抑制剂三甲基嘌呤组(3-MA组)在细胞培养液中加入加入3-MA(终浓度10 mmol/L),孵育48 h时加入LPS(终浓度1μg/ml).各组孵育4h时测定自吞噬蛋白微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)表达水平,并观察线粒体超微结构,计算自吞噬体数量,测定线粒体光密度值、线粒体膜电位(JC-1).于孵育24h时测定细胞凋亡率和Caspase-3活性.结果 与C组比较,LPS组LC3Ⅱ表达水平、线粒体光密度值、细胞凋亡率和Caspase-3活性升高,自吞噬体数量增加,JC-1降低(P＜0.05);与LPS组比较,R组LC3Ⅱ表达水平和JC-1升高,自吞噬体数量增加,线粒体光密度值、细胞凋亡率和Caspase-3活性降低,3-MA组LC3Ⅱ表达水平和JC-1降低,自吞噬体数量减少,线粒体光密度值、细胞凋亡率和Caspase-3活性升高(P＜0.05).R组线粒体超微结构损伤较LPS组减轻,3-MA组较LPS组加重.结论 自吞噬可减轻LPS诱导的HL-1心肌细胞损伤,机制可能与清除受损线粒体,改善线粒体功能,抑制细胞凋亡有关.     

Salvia przewalskii extract of total phenolic acids inhibit TLR4 signaling activation in podocyte injury induced by puromycin aminonucleoside in vitro

Background: TLR4 signaling is known to be involved in podocyte injury. We have previously shown that Salvia przewalskii extract of total phenolic acids (SPE) and its active monomer salvianolic acid B (SalB) and rosmarinic acid (RA) protect podocytes from injury induced by PAN. In the present study, we test whether SPE inhibits TLR4 signaling.

Methods: The conditionally immortalized mouse podocytes were treated with SPE, SalB, RA, SalB?+?RA or tacrolimus for 30?min, followed by PAN (100?μg/mL) for 24?h. The F-actin staining with phalloidin was used to assess cytoskeletal injury in the podocytes. Western blotting and semi-quantitatives RT-PCR were used to assess the changes of the components in the TLR4 signaling pathway.

Results: (1) The F-actin stress fibers of podocytes were almost completely disrupted after PAN treatment for 24?h, and the disruption was significantly alleviated by SPE; (2) the PAN-induced elevation of mRNA levels of TLR4, MyD88 and p65 were inhibited except p65 with high-dose SalB; (3) consistently, the protein levels of TLR4, MyD88 and pp65 were significantly elevated by PAN, and SPE, SalB, RA and admixture, respectively, attenuated the elevations of TLR4 and pp65 proteins; (4) SPE and tacrolimus have a similarly strong effect on inhibition of the expression of TLR4 signaling components.

Conclusions: SPE protects podocytes from PAN-induced injury at least partly through inhibiting TLR4 signaling. SPE is as strong as tacrolimus in inhibiting TLR4 signaling in podocytes.     

The influence of serum 25(OH)D levels on abnormal glucose metabolism induced by glucocorticoid in renal glomerular disease patients

Objective To explore the levels of serum 25(OH)D in glomerular disease patients and investigate its influence on the impaired glucose metabolism after treated with glucocorticoid. Methods A total of 61 patients with glomerular disease confirmed by clinical diagnosis and renal biopsy were included in the case group before receiving steroid therapy. 16 cases were selected as control at the same period. Before and six weeks after the treatment of glucocorticoid, all subjects took oral glucose tolerance test (OGT). According to the results of OGT, patients were divided into normal glucose regulation (NGR) group, impaired glucose regulation (IGR) group and steroid diabetes mellitus(SDM) group. Serum 25(OH)D levels were detected with enzyme-linked immunosorbent assay (ELISA), and other clinical data including albumin(Alb), Scr and urine protein were collected. Results (1) Before treated with glucocorticoid, the serum 25(OH)D levels in the control group [(64.09±13.53) nmol/L] were significantly higher than that in NGR group [(50.81±12.44) nmol/L], while the latter was significantly higher than that in IGR group [(42.71±8.09) nmol/L, all P＜0.05]. In the glomerular disease patients, 18 cases (29.51%) were 25(OH)D insufficiency and 39 cases(63.93%) were 25(OH)D deficiency. 25(OH)D levels in patients with nephritis were significantly higher than in patients with nephroitic syndrome[(56.94±10.41) nmol/L vs (45.88±11.55) nmol/L, P＜0.05]. (2)6 weeks after the treatment, incidence of steroid diabetes in IGR group was significantly higher than that in NGR group (61.11% vs 20.93%, P＜0.05). Baseline levels of 25(OH)D in NGR group and IGR group were all significantly higher than that in SDM group [(55.68±13.09) nmol/L, (48.97±9.91) nmol/L vs (40.91±7.82) nmol/L, all P＜0.05]. (3)Serum levels of 25(OH)D were positively correlated with serum albumin and serum calcium, and negatively correlated with BMI, urinary protein, cholesterol and the 2 hours postprandial blood glucose. (4)Logistic regression analysis indicated that 25(OH)D＜50 nmol/L and HbA1c＞5.6% increased the risk of developing steroid diabetes 5.586 and 5.197 times, respectively. Age increased 10 years or insulin resistance index increased one, the risk of occurred SDM increased 2.443 and 2.755 times, respectively. Conclusions Most glomerular disease patients are serum 25(OH)D deficiency or insufficiency. Low level of serum 25(OH)D is one of the main risk factors of steroid diabetes in patients with glomerular disease when treated with glucocorticoid.     

Role of ROCK1 in the podocyte injury induced by oxidized low-density lipoprotein

Objective To explore the role of ROCK1 in oxidized low-density lipoprotein (ox-LDL) induced podocyte injury and its possible mechanism. Methods The conditionally immortalized mouse podocyte cells were cultured in vitro and exposed to 20 μg/ml ox-LDL for 24 h. Western blotting was used to analyze the expression level of p-MYPT, nephrin, LC3-Ⅱ, p62, p-ULK1 in groups of control, ox-LDL, ROCK1 siRNA with ox-LDL, wtROCK1 with ox-LDL. Podocytes were incubated with DiI labeled ox-LDL for 4 h and fluorescence microscope was used to analyze lipid distribution. Results Compared with control group, ox-LDL increased cell cholesterol accumulation, activated ROCK along with decreased nephrin, LC3-Ⅱ(P＜0.05), and increased p62, and p-ULK1 expression (P＜0.05). Over-expression of ROCK1 significantly decreased the expression of nephrin and LC3-Ⅱ, but up-regulated the levels of p62, p-ULK1 and cell cholesterol accumulation in ox-LDL stimulated podocytes (P＜0.05). In contrast, Inhibition of ROCK1 protected podocyte by improved lipophagy. Conclusion ROCK1 mediated disfunction of lipophagy contributes to the ox-LDL induced podocyte injury.     

红细胞生成素对高糖诱导肾小管细胞凋亡的影响

目的 探讨红细胞生成素（EPO）是否可以抑制高糖诱导的大鼠近端肾小管上皮细胞凋亡及其相关机制。 方法 传代培养大鼠近端肾小管上皮细胞（NRK-52E）,分为正常对照组（NC组）、渗透浓度对照组（OC组）、高糖组（HG组）、高糖+EPO 50 U/ml组（E1组）和高糖+EPO 100 U/ml组（E2组）。免疫荧光检测NRK-52E细胞有无EPO受体（EPOR）表达。Western印迹检测高糖对EPOR表达的影响。流式细胞仪Annexin V-FITC/PI双染法检测细胞凋亡指数。荧光探针CM-H2DCFDA检测细胞内活性氧（ROS）的水平。RT-PCR检测bcl-2、bax、capases-3 mRNA的表达。 结果 （1）NRK-52E细胞表达EPOR,且高糖可刺激EPOR表达增加。（2）高糖可诱导NRK-52E细胞凋亡,与葡萄糖相同渗透浓度的甘露醇不能明显诱导细胞凋亡。E1、E2组细胞早、晚期凋亡率显著低于HG组（P < 0.05）。（3）高糖刺激 NRK-52E细胞后,细胞内ROS产生增多,bcl-2 mRNA的表达下调,bax、caspase-3 mRNA的表达上调。EPO可以抑制细胞内ROS的产生,上调bcl-2 mRNA 表达,下调bax、caspase-3 mRNA 表达。 结论 EPO可能通过EPOR的介导,缓解高糖诱导的氧化应激,上调bcl-2 mRNA表达,下调bax、caspase-3 mRNA表达,抑制NRK-52E细胞凋亡。     

自噬在结核菌素诱导破骨细胞形成中的作用及机制

目的 研究自噬在结核菌素纯化蛋白洐生物(PPD)诱导破骨细胞形成中的作用及机制.方法 培养小鼠单核巨噬细胞(RAW264.7细胞),用不同浓度(1.0、2.5、5.0、10.0 IU/mL)PPD及自噬激动剂雷帕霉素、自噬抑制剂3-甲基腺苷(3-MA)处理.处理24 h后,检测细胞活力;处理7 d后,检测破骨细胞数量、...