cAMP/PKA信号通路介导人脂肪干细胞成骨分化的体外试验

目的 研究环腺苷酸(cAMP)/蛋白激酶A(PKA)信号通路对脂肪干细胞(ADSCs)成骨分化的影响.方法 在成骨细胞培养基中加入cAMP/PKA信号通路的激动剂db-cAMP和抑制剂H-89,观察成骨细胞分化、PKA的活化及下游调节蛋白CREB的磷酸化和成骨基因表达的变化.结果 db-cAMP促进PKA的活化,上调CREB的磷酸化和相关基因表达的变化,促进ADSCs的成骨分化,同时以上作用可被PKA抑制剂H-89有效地阻断.结论 cAMP/PKA是介导人ADSCs成骨分化的重要信号通路.     

尿毒症毒素硫酸吲哚酚在体外对人肾小球足细胞骨架的损伤及其机理研究

目的硫酸吲哚酚(IS)是一种公认的结合蛋白质的肠源性尿毒症毒素,在慢性肾脏病患者体内蓄积可导致和促进慢性肾脏病的发生和发展。本实验探索了IS是否能引起人肾小球足细胞骨架的损伤及其可能的机理。 方法以体外培养的人肾小球足细胞系作为研究对象。采用台盼蓝拒染法、MTT法检测足细胞活力;采用免疫荧光法检测足细胞骨架纤维状肌动蛋白(F－actin)、骨架蛋白Synaptopodin改变;Western印迹法分析骨架蛋白Synaptopodin蛋白水平;RT－qPCR法检测Synaptopodin mRNA表达;琼脂糖凝胶电泳法分析足细胞内蛋白激酶A(PKA)活性。 结果IS使足细胞F－actin、Synaptopodin蛋白荧光减弱;IS还下调Synaptopodin的mRNA (P<0.01)。IS刺激使PKA磷酸化增多;PKA通路阻断剂H89可以上调Synaptopodin的蛋白及mRNA表达(P＝0.002)。 结论IS可下调足细胞骨架F－actin、Synaptopodin表达,PKA信号通路可能部分参与了Synaptopodin表达的调节,本研究结果为防治和干预CKD发生发展提供了新的思路和潜在的治疗靶点。     

血管生成素的异常表达与大鼠肾小球毛细血管损失的关系及其意义

目的 探讨足细胞损伤后，肾小球内血管生成素（Ang）1和Ang-2的表达改变与肾小球毛细血管丧失的关系及其意义。方法 100只健康雄性Wistar大鼠，随机分为假手术（Sham）组30只、单侧肾切除（UNX）组30只和单侧肾切除＋柔红霉素（DRB）组加只。DRB组大鼠，切除左肾后的第7、14天，从尾静脉各注射柔红霉素5mg／kg 1次。Sham组和UNX组亦同时以等量生理盐水尾静脉注射。完成上述处理后的第1、2、4、6、8周，随机取各组大鼠6只，采血和24h尿液检测Ccr。用PAS染色、免疫组化和原位杂交进行肾组织学分析，并用TUNEL法和透射电镜检测细胞凋亡。结果 与Sham组及UNX组比较，DRB组的Ang-1 mRNA和蛋白表达量呈显著下降的趋势；Ang-2 mRNA和蛋白表达量呈逐渐增高的趋势；Fas、FasL和caspase-3蛋白在肾小球内的表达也呈逐渐增高的趋势；肾小球凋亡指数（GAI）和肾小球硬化指数（GSI）呈逐步增高的趋势，而肾小球毛细血管密度（GCD）和Ccr呈逐步下降的趋势；透射电镜下可见到凋亡的内皮细胞。相关分析显示，Ang-1 mRNA和蛋白分别与Fas、FasL和caspase-3蛋白表达呈负相关，与GCD、Ccr呈正相关，与GAI、GSI呈负相关。Ang-2 mRNA和蛋白分别与Fas、FasL和caspase-3蛋白表达呈正相关，与GCD、Ccr呈负相关，与GAI、GSI呈正相关。结论 肾小球足细胞损伤后，局部Ang-1和Ang-2表达的平衡发生改变。这种变化与Fas／FasL及caspase-3凋亡途径的活化相关，并可能通过促进肾小球内皮细胞的凋亡，导致肾小球毛细血管的减损，进而促进肾小球硬化的发展。     

Immunohistochemical and urinary markers of podocyte injury

Renal podocytes play an important role in glomerular filtration. We estimated podocyte injury by light microscopic examination of kidney specimens or by urinary excretion of substances related to podocytes. Fresh kidney sections from 69 patients and urinary sediments from 84 patients with various renal diseases were examined immunohistochemically using fluorescent labelling. Four kinds of monoclonal antibodies which recognize podocytes were used: (1) anti-podocalyxin (anti-PCX) antibody, (2) anti-C3b receptor (anti-C3bR) antibody, (3) anti-podocyte protein, 44 kilodalton, (anti-pp44) antibody, and (4) anti-alpha 3 integrin (anti-Intα3) antibody. Labelling of kidney sections by anti-C3bR (k-C3bR) was reduced in cases of severe glomerular injury, although there were no changes in k-PCX, k-pp44, or k-Intα3 labelling. PCX labelling of urinary sediments (u-PCX) was detected in nearly all cases of glomerulonephritis, u-C3bR was seen in some cases of glomerular injury, u-Intα3 was seen in only a small number of cases of severe glomerular injury, and u-pp44 was not detected in any urinary samples. u-PCX, u-C3bR, u-Intα3, and k-C3bR correlated with the degree of pathological change, the degree of proteinuria and hematuria. These results suggest that immunostaining kidney sections with anti-C3bR antibody and urinary sediments with anti-PCX, anti-C3bR, and anti-Intα3 antibodies might be useful in detecting podocyte injury. Received March 17, 1997; received in revised form and accepted August 1, 1997     

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.     

IgA肾病患者血清IgA1与系膜细胞共培养上清诱导足细胞凋亡

目的 探讨IgA肾病(IgAN)患者血清IgA1与系膜细胞共培养上清对足细胞凋亡的影响。 方法 用Jacalin 亲和层析柱和Sephacryl S-200 分子筛纯化蛋白。单体IgA1（mIgA1）热聚合为聚合体IgA1（aIgA1）。实验分为患者上清组、健康上清组和对照组，系膜细胞分别与IgAN患者的aIgA1、健康对照的aIgA1和5%胎牛血清共培养，收集上清，与同步化的足细胞作用。流式细胞仪检测细胞凋亡情况。实时定量PCR 检测凋亡相关基因Bcl-2、Bax、Fas和Fas-L表达情况。 结果 患者上清可诱导足细胞凋亡，其凋亡率显著高于健康上清组和对照组[（28.5±5.9）%比（22.5±5.8）%、（20.5±4.5）%， 均P < 0.05]。患者上清可诱导足细胞Fas mRNA 升高，为对照组的1.89倍（P < 0.05）， 而Bcl-2 mRNA下调为对照组的72%（P < 0.05）。患者上清组的AngⅡ和TGF-β1水平均高于健康上清组[（13.2±3.4） ng/L比（8.2±2.3） ng/L，P < 0.05；（15.4±3.4） ng/L比（10.8±3.2） ng/L，P < 0.05]。 结论 IgAN患者血清IgA1与系膜细胞共培养上清可诱导足细胞凋亡，可能参与IgAN的进展。     

血管紧张素Ⅱ灌注诱导nephrin表达改变与足细胞凋亡

目的 研究血管紧张素Ⅱ（AngⅡ）灌注对大鼠足细胞裂隙膜分子nephrin表达及足细胞凋亡的影响，以及探讨AngⅡ引起蛋白尿及肾小球硬化的机制。方法 36只雄性Sprague Dawley大鼠分为AngⅡ灌注组(400 ng&#8226;kg-1&#8226;min-1)、生理盐水灌注组和正常对照组，测定28 d内大鼠血压及尿蛋白。分别于14、28 d处死动物取肾，观察组织学改变，并用免疫荧光、免疫电镜检测nephrin分布。RT-PCR及Western印迹法分别检测nephrin mRNA及蛋白表达。TUNEL法检测足细胞凋亡。结果 (1) AngⅡ灌注组大鼠血压升高，14 d达峰值并维持该水平至28 d；AngⅡ灌注7 d即出现蛋白尿，并持续增加。(2) AngⅡ灌注14 d时，足细胞裂隙膜变窄；灌注28 d时，足突增宽及节段性融合，部分足细胞有凋亡小体形成，少数肾小球出现节段性硬化。TUNEL法检测发现足细胞凋亡[（2.7±1.6）个/肾小球切面]，凋亡数与蛋白尿量呈正相关（r = 0.86，P < 0.01）。(3) AngⅡ灌注14 d时，肾皮质nephrin mRNA及蛋白表达上调（P < 0.05）。nephrin由正常的沿毛细血管袢线状分布向粗颗粒、团块状分布模式转变。AngⅡ灌注28 d时，肾皮质nephrin mRNA及蛋白表达下降（P < 0.05），且nephrin蛋白表达与足细胞凋亡数呈负相关（r = －0.63，P < 0.01）。 结论 AngⅡ灌注诱导的nephrin表达及分布改变可能导致了足细胞凋亡及肾小球硬化的发生与发展。     

Tacrolimus restores podocyte injury and stabilizes the expression of Cabin1 in 5/6 nephrectomized rats

Podocyte injury is a vital factor, which induces massive proteinuria. Studies have shown that tacrolimus (TAC) protected podocyte via stabilizing cytoskeleton. Our latest study indicates that calcineurin binding protein 1 (Cabin1) undergoes nuclear translocation during podocytes injury. Whether TAC targets on Cabin1 during podocyte injury is still not clear. This study establishes non-immunological proteinuric model. To observe the effect of the treatment of TAC on Cabin1 expression in 5/6 nephrectomized rats. Sprague–Dawley rats were injected with TAC (0.2?mg/kg/day) for 4–8 weeks after 5/6 nephrectomy. Then, rats were sacrificed in the eighth week after operation, renal tissues were processed for morphological studies under light and electrical microscope. Cabin1 expression and distribution were detected by western blot and indirect immunofluorescence staining. In 5/6 nephrectomized rats, urinary protein excretion reached 90.2?±?30.1?mg/24?h, glomerular sclerosis index and tubulointerstitial fibrosis score were significantly increased, and widespread of podocyte foot processes fusion was found. Moreover, Cabin1 protein expression was markedly increased, and its distribution became much more obviously in podocytes nuclei. In TAC treated rats, urinary protein excretion significantly decreased (44.9?±?22.5?mg/24?h), glomerular sclerosis and tubulointerstitial fibrosis were alleviated, and podocyte foot processes fusion was inhibited. Furthermore, TAC alleviated the increased protein expression and abnormal distribution of Cabin1. In conclusion, TAC restores podocyte injury and stabilizes the expression of Cabin1. Cabin1 may become a new target to demonstrate the mechanism of TAC in podocyte injury.     

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。     

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。     

蛋白激酶A活性下降对膀胱移行细胞癌凋亡的影响

目的探讨蛋白激酶A(PKA)活性下降对膀胱移行细胞癌细胞株T24增殖和化疗敏感性的影响及其机制。方法用0、15、30μmol/L H-89(PKA活性抑制剂)诱导T24细胞后,用放射免疫法检测H-89作用下T24细胞中PKA活性的变化；用流式细胞仪检测H-89作用下细胞凋亡的变化；用噻唑蓝(MTT)比色法检测丝裂霉素和表阿霉素单独作用下以及与15μmol/L H-89共同作用下T24细胞活力的变化；用Western blot检测15μmol/L H-89作用下T24细胞中bcl-2表达的变化。结果H-89能够使T24细胞中的PKA活性下降；在15μmol/LH-89作用36h后,流式细胞仪检测可见T24细胞凋亡率为57．81%,显著高于对照组4．37%(F=311．35,P＜0．01)。丝裂霉素(MMC)和表阿霉素(Epirubicin)对T24细胞的半数致死量分别为120mg/L和100mg/L；加入15μmol/L H-89后,细胞的存活率分别降为(31．14±4．37)%和(24．91±3．59)%,两组间差异有统计学意义(丝裂霉素组：F=23．94,P＜0．01；表阿霉素组：F=50．64,P＜0．01)。用15μmol/L H-89诱导T24细胞后,bcl-2表达呈下降趋势,并显著低于对照组。结论抑制PKA活性能够使bcl-2的表达下降,从而诱导膀胱癌T24细胞株凋亡,并提高其对化疗药物的敏感性。     

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.     

他克莫司对阿霉素肾病大鼠足细胞损伤的修复作用

目的探讨他克莫司对阿霉素肾病大鼠足细胞损伤的修复作用。方法通过阿霉素尾静脉注射建立大鼠微小病变肾病模型,并以他克莫司进行干预。大鼠随机分为对照组、模型组和他克莫司组。每组分别于第0、7、14、21、28、35天采集尿液,检测尿蛋白排泄量,于造模第35天处死大鼠收集肾脏标本。电镜观察各组大鼠足细胞足突融合情况;TUNEL检测法对肾小球进行染色;免疫组化或免疫荧光对WT-1、caspase-3进行定位和半定量检测,观察大鼠足细胞的数量和凋亡。结果阿霉素尾静脉注射后大鼠尿蛋白显著增加,足细胞数目明显减少,足突广泛融合;与模型组比较,他克莫司组大鼠24 h尿蛋白明显减少,足突融合改善,足细胞数目增多。与对照组比较,模型组大鼠肾小球内caspase-3蛋白表达增加,TUNEL染色加深;与模型组比较,他克莫司组肾小球内caspase-3表达量下降,TUNEL阳性率减少,说明足细胞凋亡减少。结论他克莫司可修复阿霉素肾病大鼠足细胞损伤,其机制与抑制足细胞凋亡有关。     

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.     

霉酚酸酯及缬沙坦对糖尿病大鼠足细胞的保护机制研究

目的探讨霉酚酸酯、缬沙坦及2者联合应用对糖尿病。肾病（DN）大鼠足细胞损伤的保护作用。方法雄性Wistar大鼠行右肾切除后，腹腔注射链脲佐菌素（STZ，65mg／kg）建立糖尿病模型。将实验动物随机分为右。肾切除对照组（NC）、糖尿病组（DM）、霉酚酸酯治疗组（M）、缬沙坦治疗组（V）、缬沙坦和霉酚酸酯联合治疗组（V＋M）。治疗组分别给予霉酚酸酯15mg·kg^-1·d^-1，缬沙坦40mg·kg^-1·d^-1；联合治疗组为上述两组之和。检测各组8周末的左肾质量／体质量比值、尿蛋白量（24h）、血糖（Glu）、Scr。光镜及电镜观察肾组织形态学变化。免疫组化检测肾组织中nephrin、结蛋白（desmin）及单核细胞趋化因子1（MCP-1）蛋白表达。实时PCR测定肾组织中nephrin及MCP-1mRNA表达。结果与NC组相比，DM组大鼠血糖、尿蛋白量及左肾质量／体质量比值均显著上升（P〈0．01）；肾小球硬化指数（GSI）及肾间质损害加重（P〈0．01）；肾组织内MCP-1、desmin蛋白表达均显著上调（P〈0．01）。与DM组比较，M组、V组及V＋M组上述指标除Glu、Scr外，均明显改善（P〈0．05或P〈0．01）。与NC组（100％）相比，DM组nephrinmRNA表达下调（78％，P〈0.05）；各治疗组nephrinmRNA表达增加，以M组增加最明显（134％，P〈0．01）。与NC组（100％）相比，DM组MCP-1mRNA表达明显上调（251％，P〈0.05）；各治疗组明显降低，以M组最显著（126％，P〈0．01）。nephrinmRNA与MCP-1mRNA表达呈负相关（r=-0，86。P〈0．01）。尿蛋白量（24h）与MCP-1mRNA呈正相关fr=0．82，P〈0．01）；与nephrinmRNA呈负相关（r=-0．78，P〈0．01）。结论霉酚酸酯及缬沙坦均能下调糖尿病大鼠肾组织中desmin及MCP-1基因及蛋白的表达，上调nephrin基因及蛋白表达，降低尿蛋白量，预防肾损伤。联合治疗不优于单一治疗。霉酚酸酯可能通过抗炎性反应减轻足细胞损伤，减少蛋白尿，对早期DN大鼠具有明显的肾保护作用。