高糖通过PI3K-AKT-mTOR信号通路增加足细胞自噬

目的 研究高糖引起足细胞自噬变化及其相关的信号机制.方法 培养的足细胞被分为6组,正常浓度葡萄糖(NG)组、高浓度葡萄糖(HG)组、NG+雷帕霉素(Rap)组、HG+Rap组、NG+LY294002组和HG+LY294002组.观察自噬增强剂Rap和PI3K抑制剂LY294002对高糖条件下培养的足细胞自噬和凋亡的影响.电镜和吖啶橙染色观察细胞内自噬体的形成;Western印迹检测自噬标志蛋白微管相关蛋白1轻链3(LC3)和自噬血管基因Beclin-1的表达;通过阻断自噬的信号通路观察磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)相关蛋白AKT和mTOR的磷酸化水平的改变.结果 高糖可导致足细胞凋亡增加,促进足细胞内自噬体和自噬相关蛋白表达增加(均P＜ 0.05).与高糖组相比,HG+ Rap组LC3-Ⅱ和Beclin-1的表达增加(均P＜0.05);LY294002部分抑制高糖导致的LC3-Ⅱ和Beclin-1表达增加(均P＜0.05).与高糖组相比,HG+ LY294002组足细胞内AKT磷酸化的水平增加(P＜0.05),mTOR的磷酸化水平降低(P＜0.01);HG+ LY294002组足细胞的AKT和mTOR磷酸化水平较高糖组均降低(均P＜0.05).结论 高糖可促进足细胞的自噬和凋亡,推测高糖诱导的足细胞自噬作用部分通过PI3K-AKT-mTOR信号通路调节实现的.     

番茄红素对高糖诱导的足细胞自噬以及凋亡的影响

目的探讨番茄红素(Lyc)对高糖诱导的足细胞自噬及凋亡的影响,旨在为临床治疗糖尿病肾病提供可靠的理论依据。方法采用条件永生化小鼠足细胞系为研究对象,通过添加不同剂量的Lyc对高糖诱导的足细胞进行干预。实验细胞分6组,分别为正常对照组(5.5m m ol/L葡萄糖)、高渗对照组(5.5 m m ol/L葡萄糖+1 9.5 m m ol/L甘露醇)、高糖组(2 5 m m ol/L葡萄糖)、低剂量Lyc组(25 mmol/L葡萄糖+3.125μmol/L Lyc)、中剂量Lyc组(25 mmol/L葡萄糖+6.25μmol/L Lyc)和高剂量Lyc组(25 mmol/L葡萄糖+12.5μmol/L Lyc)。在细胞培养4 8 h后,用吖啶橙染色法观察各组细胞的自噬体变化情况;流式细胞技术检测各组细胞的凋亡率变化;采用实时定量荧光PCR和蛋白质印迹法分别检测自噬相关基因LC3、beclin-1、p62/SQSTM I m RNA和蛋白的表达变化情况。结果与正常对照组、高渗对照组相比,高糖组细胞中的自噬体数目明显增多,细胞的凋亡率极显著增加(P0.01),自噬相关基因LC3-II、beclin-1的表达量显著增加(P0.05),而p62/SQSTMI基因的表达量则显著降低(P0.05)。与高糖组比较,不同剂量Lyc组中的足细胞自噬体数目更多;细胞的凋亡率显著降低(P0.05),且高剂量Lyc组细胞的凋亡率降低极显著(P0.01);自噬相关基因LC3-II、beclin-1表达量显著增加(P0.05);而p62/SQSTMI基因表达量则显著降低(P0.05);且高剂量Lyc组基因表达量变化极显著(P0.01);蛋白水平上的检测结果与mRNA水平基本一致。结论Lyc能进一步增强高糖诱导引起的足细胞的自噬,但对足细胞的凋亡却起到了一定的抑制作用,且高剂量的Lyc作用效果更加明显。     

疏利分消方调控自噬对补体攻膜复合物所致足细胞损伤的内在机制研究

目的:以补体攻膜复合物诱导足细胞损伤体外模拟特发性膜性肾病发病过程,探讨疏利分消方冻干粉对自噬通路的调控作用.方法:以酵母多糖活化血清(ZAS)体外组装C5b-9,并建立C5b-9损伤足细胞模型,设置正常组、模型组和模型+中药组,检测各组乳酸脱氢酶(LDH)释放率、自噬标记蛋白LC3及自噬底物蛋白P62荧光表达.结果:...     

天麻素通过调控Klotho基因表达对高糖诱导的足细胞损伤的影响

目的:探讨天麻素对高糖诱导的足细胞损伤的影响及分子机制。方法:将小鼠肾脏足细胞MPC5分为对照组、高糖组、高糖+天麻素低、中、高剂量组、高糖+pcDNA组、高糖+pcDNA-Klotho组、高糖+天麻素+si-NC组、高糖+天麻素+si-Klotho组。蛋白质印迹(Western blot)法检测蛋白表达;流式细胞术检测细胞凋亡;实时荧光定量PCR(RT-qPCR)检测Klotho mRNA表达水平。结果:天麻素低、中、高剂量处理后,高糖诱导的足细胞中nephrin和podocin蛋白表达水平升高,细胞凋亡率降低,Bcl-2蛋白表达水平升高,Bax蛋白表达水平降低,Klotho mRNA和蛋白表达水平升高,且呈剂量依赖性(P0.05)。过表达Klotho可抑制高糖诱导的足细胞凋亡,提高nephrin、podocin蛋白表达水平。抑制Klotho表达逆转了天麻素对高糖诱导的小鼠足细胞损伤的保护作用。结论:天麻素通过上调Klotho表达抑制高糖诱导的足细胞损伤。     

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。     

氯喹抑制高糖环境下前列腺癌PC3细胞增殖机制研究

目的:研究氯喹是否能通过自噬和活性氧(ROS)通路抑制高糖诱导的前列腺癌PC3细胞的增殖。方法:PC3细胞培养后分为4组(正常糖组、高糖组、正常糖+氯喹组、高糖+氯喹组)予相应干预。运用CCK-8实验检测细胞的增殖能力;Western blot检测自噬相关蛋白LC3的表达;DCFH-DA活性氧荧光探针检测细胞中ROS水平;JC-1法检测细胞线粒体膜电位的去极化。结果:CCK-8实验结果表明氯喹能够显著抑制高糖环境下PC3细胞的增殖。Western blot实验结果显示氯喹会使PC3细胞LC3-Ⅱ/Ⅰ比值升高,说明细胞自噬显著被抑制。DCFH-DA探针检测结果表明,氯喹能够显著上调高糖环境下PC3细胞中ROS水平。JC-1检测结果表明,氯喹能够诱导高糖环境下PC3细胞中线粒体膜电位去极化。结论:氯喹通过自噬和ROS通路抑制高糖环境下前列腺癌细胞的增殖。     

益肾胶囊含药血清对高糖环境下肾小球足细胞自噬的影响

目的:观察益肾胶囊含药血清对高糖环境下小鼠肾小球足细胞自噬的影响。方法:制备不同浓度益肾胶囊含药血清,对体外培养的小鼠肾小球足细胞进行分组干预,细胞分为:正常组(NG)、高糖组(HG)、低浓度益肾胶囊含药血清组(LY)、中浓度益肾胶囊含药血清组(MY)、高浓度益肾胶囊含药血清组(HY)、贝那普利含药血清组(BP)。Western Blot检测自噬相关LC3蛋白及泛素结合蛋白p62(p62/SQSTMl)蛋白的表达变化。透射电镜下观察各组足细胞内自噬体的变化。结果:与NG组相比,HG组足细胞自噬相关蛋白LC3Ⅱ表达减少,泛素结合蛋白p62(p62/SQSTMl)表达增多(P0.05);与HG组相比,HY组足细胞自噬相关蛋白LC3Ⅱ表达增多,泛素结合蛋白p62(p62/SQSTMl)表达减少(P0.05)。透射电镜提示,与HG组相比,HY组足细胞自噬体增多(P0.05)。结论:高糖可导致足细胞自噬障碍,而益肾胶囊可通过改善其自噬障碍,进而发挥足细胞保护的作用。     

陈氏益气活血化湿方通过调控足细胞自噬减轻PAN诱导的足细胞损伤

目的:本研究旨在观察陈氏益气活血化湿方水溶剂对氨基核苷嘌呤霉素(puromycin aminonucleoside,PAN)诱导足细胞损伤的保护作用,并探讨这一作用与足细胞自噬(autophagy)的关系。方法:体外培养永生化小鼠足细胞系作为研究对象,采用细胞增殖检测试剂盒(CCK-8法)、western bloting法检测不同浓度PAN对足细胞的存活率的影响,建立PAN诱导的足细胞损伤模型。后将足细胞分为:正常组、PAN组、PAN+中药低剂量组、PAN+中药高剂量组,采用western bloting法检测足细胞p-mTOR、mTOR、LC3、synaptopodin蛋白的表达情况。结果:(1) CCK-8、western bloting检测结果显示50μg/ml浓度时P-mTOR表达最明显(P 0. 001),确定为造模浓度;(2) PAN造成足细胞p-mTOR表达升高,LC3II、synaptopodin蛋白表达降低(P 0. 05),mTOR磷酸化水平升高,足细胞自噬功能受到抑制,足细胞骨架蛋白表达降低(P 0. 01),足细胞受损;(3)陈氏益气活血化湿方水溶剂处理细胞后p-mTOR表达降低,LC3II、synaptopodin蛋白表达升高(P 0. 05),且高剂量组效果优于低剂量组。结论:陈氏益气活血化湿方水溶剂可能是通过上调足细胞自噬功能,发挥足细胞保护作用。     

狼疮肾炎患者自噬水平及其对足细胞相关蛋白表达水平的影响

目的探讨狼疮肾炎(lupus nephritis,LN)患者自噬水平及其对足细胞相关蛋白表达水平的影响。方法选择2017年5月至2019年5月于榆林市第一医院收治的69例LN患者为LN组,50例系统性红斑狼疮(systemic lupus erythematosus,SLE)患者为SLE组,50例肾切除手术患者为对照组,观察3组肾脏足细胞内自噬体数量,比较3组肾脏组织中自噬相关蛋白、微管相关蛋白轻链3(LC3)、B淋巴细胞瘤蛋白质-2-相互作用蛋白(Beclin1)的表达,以及足细胞相关蛋白,肾病蛋白(Nephrin)、足突蛋白(Podocin)的表达。分离狼疮肾炎患者肾脏足细胞,将足细胞分为自噬抑制组和自噬诱导组,自噬抑制组加入100 nmol/L 3-甲基腺嘌呤(3-MA),自噬诱导组加入100 nmol/L雷帕霉素(RAPA),比较3组足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin等蛋白的表达。结果LN组、SLE组肾脏足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin蛋白表达量显著高于对照组(P<0.05);SLE组肾脏足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin蛋白表达量显著高于LN组(P<0.05);自噬诱导组足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin蛋白表达量显著高于正常对照组、自噬抑制组(P<0.05);正常对照组足细胞内自噬体数量及LC3、Beclin-1、Podocin、Nephrin蛋白表达量显著高于自噬抑制组(P<0.05)。结论LN患者自噬水平呈升高状态,自噬水平升高可能通过上调足细胞相关蛋白Podocin、Nephrin水平而减轻足细胞损伤,抑制LN病情进展。     

三氧化二砷诱导肝癌细胞系HepG-2自噬及其机制研究

目的:测定不同浓度三氧化二砷(As2O3)诱导前后HepG-2细胞自噬水平改变,并初步探讨机制。方法:常规细胞培养,按As2O3不同浓度分组;采用MDC染色,荧光显微镜观察自噬囊泡,流式细胞术检测其荧光强度;RT-PCR检测Bcl-2基因转录,免疫组织化学检测细胞Bcl-2蛋白阳性百分率,并分析同自噬的关系。结果:经不同浓度As2O3作用后,HepG-2细胞生长明显受到抑制;荧光显微镜可观察到自噬囊泡的出现;Bcl-2基因表达降低;经相关性分析,HL-60细胞自噬水平与Bcl-2基因表达呈负相关。结论:As2O3可诱导HepG-2细胞自噬,v诱导HepG-2细胞自噬的机制可能与下调Bcl-2基因表达有关,自噬和凋亡存在交叉。     

Human umbilical cord mesenchymal stem cells co-culture ameliorates the innate immunity of podocytes induced by high glucose

Objective To explore the effects of human umbilical cord mesenchymal stem cells(HUC-MSCs) on the innate immunity of podocytes mediated by Toll-like receptor (TLR) signaling pathway under high glucose (HG) condition. Methods Podocytes were divided into four groups according to the treatment: normal glucose group (NG), mannitol control group (NG+MA), high glucose group (HG) and HUC-MSCs co-culture group (HG+MSC). After 72 hours treatment, the protein levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), heat shock protein 70 (HSP70), high-mobility group box-1 (HMGB1) in culture medium were measured by ELISA. Real-time PCR was used to detect the mRNA expressions of TLR2 and TLR4. Western blotting was used to detect the protein expressions of TLR2, TLR4, myeloid differentiation factor 88 (MyD-88) and phospho-P65 (p-P65). Immunofluorescence staining was used to study the localization of p-P65 in podocytes. Results High glucose induced the inflammation of podocytes by activating the TLR signaling, which increased the secretion of IL-6, TNF-α, HSP70, HMGB1, the mRNA level of TLR2, TLR4 and the protein level of TLR2, TLR4, MyD-88 and p-P65 (all P＜0.05). High glucose also activated NF-κB and induced its nuclear translocation. HUC-MSCs co-culture decreased the inflammation and restrained the TLR signaling. Conclusions HUC-MSCs co-culture decreases the inflammation and innate immunity of podocytes induced by HG.     

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.     

Role of podocyte autophagy in passive Heymann nephritis

Objective To investigate the role of autophagy in podocyte damage, and the intracellular mechanism of autophagy activation through passive Heymann nephritis (PHN) animal model. Methods Male Sprague-Dawley rats (n=40) were studied on day 0, 2, 4, 7, and 21 after induction of PHN by injection of anti-Fx1A. Podocyte morphology and autophagosomes were observed by transmission electron microscopy. Podocyte numerical density was estimated by Weibel-Gomez method. Cell apoptosis was detected by TUNEL assay and caspase-3 immunohistochemical staining. Expressions of autophagy markers and endoplasmic reticulum stress (ERS)-associated proteins were analyzed by Western blotting. Results (1) In PHN rats, immunohistochemical staining showed that C5b-9 deposited along glomerular basement membrane on day 4 to day 21. Small subepithelial electron-dense deposits and a part of foot process fusion were detected in the glomerulus of PHN rats on day 4 by transmission electron microscope, and podocyte damage was aggravated on day 21. Furthermore, compared with control, the urinary protein levels of PHN rats began to increase on day 3, and reached the top on day 21 [(50.6±6.0) mg/24 h]. (2) The number of podocytes significantly decreased in PHN rats compared with control group on day 21(P＜0.05). (3) In PHN rats, apoptotic podocytes were found by caspase-3 immunohistochemical staining and TUNEL assay on day 21. (4) The expression of autophagy marker LC3Ⅱwas markedly increased on day 7 and 21, but down-regulated on day 21 compared with day 7. Moreover, accumulated autophagosomes in podocytes were detected on day 7 and 21 by transmission electron microscope. (5) The level of GRP78 was significantly increased on day 2 and 7 but reduced to baseline on day 21. At the same time, the downstream pathways (ATF6α, p-PERK and p-JNK) of unfolded protein response were also up-regulated in the early process of PHN and down-regulated later. Conclusions Autophagy is an important way to protect against immune-mediated podocyte injury in membranous nephropathy. Autophagy activation is mainly related to endoplasmic reticulum stress induced by complement attack. This provides an important basis for a thorough understanding of the role of autophagy in the process of podocyte damage and the pathogenesis of membranous nephropathy.     

表没食子儿茶素没食子酸酯对高糖环境下体外小鼠足细胞活性氧的影响

目的　观察不同浓度表没食子儿茶素没食子酸酯（EGCG）对高糖造成氧化应激状态下体外小鼠足细胞损害的作用并探讨其机制。 方法　以高糖（25 mmol/L）培养的小鼠足细胞为研究对象,维生素E培养为对照。首先以MTT法检测细胞活力,再在激光共聚焦显微镜下以CM-H2DCFDA荧光探针观察不同浓度EGCG（0.2、10、100 μmol/L）刺激足细胞6、12、24 h后活性氧（ROS）生成,并以流式细胞仪定量分析ROS平均荧光强度。RT-PCR法检测足细胞内ROS产生的主要通路NADPH氧化酶各亚基mRNA（ph22phox、p47phox、p67phox）的表达。 结果　高糖刺激下6 h,足细胞ROS生成显著增加（P < 0.01）。正常糖组和甘露醇组培养12 h ROS生成无显著增加（P > 0.05）。EGCG 0.2 μmol/L作用6 h可显著降低高糖环境下体外小鼠足细胞ROS水平（P < 0.01）。与高糖组比较,EGCG（100 μmol/L）显著减少NADPH氧化酶亚基p22phox和p67phox mRNA表达（均P < 0.05）。与维生素Ｅ组比较,EGCG（0.2 μmol/L）和维生素E（0.2 mmol/L）协同作用组显著减少p47phox mRNA表达（P < 0.05）。 结论　EGCG能缓解高糖环境下体外足细胞氧化应激状态,对高糖培养下足细胞有保护作用。     

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。     

Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes.

BACKGROUND: Recent studies show that mechanical stress modifies both morphology and protein expression in podocytes. Ambient glucose is another factor modulating protein synthesis in these cells. In diabetes, podocytes experience elevated glucose concentrations as well as mechanical strain generated by high intracapillary pressures. Both these factors are responsible for podocyte injury, leading to impairment of kidney glomerular function. In the present study, we examined the effects of glucose concentration and mechanical stress on glucose uptake in podocytes. METHODS: Following a 24 h pre-incubation in low (2.5 mM, LG), normal (5.6 mM, NG) or high (30 mM, HG) glucose media, cultured rat podocytes were exposed to 4 h mechanical stress. We used the labelled glucose analogue, [3H]2-deoxy-D-glucose, to measure glucose uptake. The distribution of facilitative glucose transporters GLUT2 and GLUT4 was assessed by flow cytometry. RESULTS: In the control (static) cells, glucose uptake was similar in the three glucose groups. In mechanically stressed podocytes, glucose uptake increased 2-fold in the LG and NG groups but increased 3-fold in the HG group. In the NG cells, mechanical load increased the membrane expression of GLUT2 and reduced the membrane-bound GLUT4. In stretched HG cells, the membrane expression of both GLUT2 and GLUT4 was decreased. High glucose decreased the plasma membrane GLUT2 content in the stretched cells, whereas both static and stretched podocytes showed an elevation in GLUT4. CONCLUSION: Mechanical stress potentiated glucose uptake in podocytes and this effect was enhanced by high ambient glucose. The decreased expression of GLUT2 and GLUT4 on the surface of stretched cells suggests that the activity of other glucose transporters may be regulated by mechanical stress in podocytes.     

Podocyte EGFR Inhibits Autophagy Through Upregulation of Rubicon in Type 2 Diabetic Nephropathy

Renal epidermal growth factor receptor (EGFR) signaling is activated in models of diabetic nephropathy (DN), and inhibition of the EGFR signaling pathway protects against the development of DN. We have now determined that in cultured podocytes, high glucose led to increases in activation of EGFR signaling but decreases in autophagy activity as indicated by decreased beclin-1 and inhibition of LC3B autophagosome formation as well as increased rubicon (an autophagy inhibitor) and SQSTM1 (autophagy substrate). Either genetic (small interfering [si]EGFR) or pharmacologic (AG1478) inhibition of EGFR signaling attenuated the decreased autophagy activity. In addition, rubicon siRNA knockdown prevented high glucose–induced inhibition of autophagy in podocytes. We further examined whether selective EGFR deletion in podocytes affected the progression of DN in type 2 diabetes. Selective podocyte EGFR deletion had no effect on body weight or fasting blood sugars in either db/db mice or nos3^−/−; db/db mice, a model of accelerated type 2 DN. However selective podocyte EGFR deletion led to relative podocyte preservation and marked reduction in albuminuria and glomerulosclerosis, renal proinflammatory cytokine/chemokine expression, and decreased profibrotic and fibrotic components in nos3^−/−; db/db mice. Podocyte EGFR deletion led to decreased podocyte expression of rubicon, in association with increased podocyte autophagy activity. Therefore, activation of EGFR signaling in podocytes contributes to progression of DN at least in part by increasing rubicon expression, leading to subsequent autophagy inhibition and podocyte injury.     

TLR4 links podocytes with the innate immune system to mediate glomerular injury

Toll-like receptors (TLR) classically recognize pathogen-associated danger signals but are also activated via endogenous ligands. For evaluation of their role in inflammatory kidney disease, the function of TLR was analyzed in two mouse models of cryoglobulinemic membranoproliferative glomerulonephritis (MPGN; mice transgenic for thymic stromal lymphopoietin [TSLP], with or without deletion of the Fcgamma receptor IIb). Expression of TLR1 through 9 and TLR11 mRNA was detectable in whole kidneys and in isolated glomeruli of wild-type mice, with TLR3 and TLR4 having the highest absolute levels of expression. TLR1, 2, and 4 were increased in TSLP transgenic mice and even higher in TSLP transgenic FcgammaRIIb-deficient mice. TLR5 through 9 and 11 were upregulated to similar degrees in TSLP transgenic and TSLP transgenic FcgammaRIIb-deficient mice. Immunohistochemical studies of nephritic glomeruli localized TLR4 protein to podocytes. Cultured podocytes also expressed TLR4, and stimulation with TLR4-specific ligands resulted in a marked induction of chemokines; this was reduced by specific knockdown of TLR4 with siRNA. Fibrinogen, a potential endogenous TLR4 ligand, was shown to induce a similar profile of chemokines. In conclusion, it was demonstrated that TLR4 is constitutively expressed by podocytes and is upregulated in MPGN, where it may mediate glomerular injury by modulating expression of chemokines; therefore, TLR4 may link podocytes with the innate immune system to mediate MPGN triggered by the deposition of immune complexes.     

Protective effect ofACTH4-10 on adriamycin-induced podocyte injury

Objective To observe the influence of adrenocorticotropic hormone (ACTH4-10) in the changes of podocyte proliferation, apoptosis and expression of nephrin and podocin on adriamycin(ADR) -induced podocyte injury and investigate the protective effect ofACTH4-10. Methods All podocytes were randomly divided into following groups: normal control, ADR-induced group andACTH4-10 intervention group (low, middle and high concentration). Normal control group was not treated, ADR-induced group was induced to set the model of podocyte injury by ADR (1 μmol/L) for 24 hours andACTH4-10 intervention groups were intervened by 1 μg/L, 10 μg/L and 100 μg/LACTH4-10 for 1 hours respectively, prior to setting the model of podocyte injury. Cell counting kit (CCK-8) was used to detect the multiplication of podocytes and TUNEL apoptosis detection kit was used to detect podocyte apoptosis. Real-time PCR and Western blotting were used to examine the expression of nephrin and podocin. Results Compared with control group, podocyte proliferation and expression of nephrin and podocin was decreased significantly in ADR-induced group (P＜0.05), meanwhile podocyte apoptosis was increased obviously (38.14% vs 5.12%). Compared with ADR-induced group, podocyte proliferation and expression of nephrin and podocin was increased generally with concentration ofACTH4-10. Although podocyte apoptosis rates (20.45%, 17.39%, 11.02%) were increased inACTH4-10 intervention group (low, middle and high concentration) while comparing with normal control group, podocyte apoptosis decreased obviously while comparing with ADR-induced group. ConclusionsACTH4-10 can stabilize the expression of nephrin and podocin on slid diaphragm, and has the protective effect on podocyte injury induced by ADR, while the effect depends on the concentration ofACTH4-10.