过敏毒素受体(C3aR)在db/db糖尿病肾病小鼠肾脏中的表达及病理意义分析

利用半定量RT-PCR、免疫组化和Western blotting的方法,同时从mRNA水平和蛋白质水平对过敏毒素受体(C3aR)在不同病理阶段的2型糖尿病肾病模型小鼠——db/db小鼠肾脏中的表达情况进行了较为系统的分析．结果发现：a．在糖尿病前的db/db小鼠(4周龄的db/db小鼠),C3aR与作为正常对照的db/m小鼠相比没有明显差异．随着肥胖的加剧,高血糖、蛋白尿的发生和发展,C3aR在db/db小鼠肾脏中的表达显著升高．b．免疫组化分析显示,C3aR广泛地表达于db/m和db/db小鼠肾脏的皮质和髓质,分布于肾脏的上皮细胞中(包括肾小管上皮细胞、肾小球中的脏层上皮细胞(足细胞)和壁层上皮细胞)．从部位来看,皮髓交界处的肾小管中C3aR表达量明显要比其他部位的多．在肾小球,C3aR特异地存在于足细胞部位．在db/m小鼠,不同周龄小鼠肾脏中C3aR的表达量并没有明显变化,但在db/db小鼠,从8周龄开始,分布在db/db小鼠肾小管上皮细胞和小球足细胞中的C3aR均随小鼠周龄的增加而增加,至少在时间上,与小鼠糖尿病肾病的发生发展相关,其中尤以足细胞中和皮髓交界处肾小管上皮细胞中的变化最为明显． c．在糖尿病肾病小鼠中高表达C3aR的肾小管上皮细胞常有空泡变性的情况．上述工作印证了先前对2型糖尿病肾病患者肾小球基因表达谱的分析结果,更加明确了C3aR与糖尿病肾病的相关性,同时揭示了C3aR在正常小鼠和糖尿病肾病小鼠肾脏中的表达、分布和变化规律,有利于进一步揭示C3aR的功能及其在糖尿病肾病发生、发展过程中的可能作用,探讨糖尿病肾病的分子机制．     

C3aR过度活化与糖尿病肾病肾组织损伤的关系

C3aR是补体C3裂解产物C3a的受体.最近的一些研究提示C3aR通路可能参与了糖尿病肾病(DN)的病理过程,但有关C3aR通路在DN中的确切病理作用及有关机制远未清楚.需要特别指出的是,现有的有关C3aR参与DN肾组织损伤的证据主要来自一些动物模型的研究,临床上尚缺乏较为系统全面的对DN患者肾组织C3aR通路与肾组织损伤关系的观察分析.为此,本文首次以较大的样本量分析了不同病理时期DN患者肾组织C3aR和C3a的表达变化情况及其与DN患者肾组织损伤的相关性.在此基础上,进而利用体外细胞模型,对高糖环境下C3aR活化致肾小球足细胞损伤的作用及机制进行了探讨.结果显示:a.与正常对照组相比,DN患者肾组织C3a和C3aR的表达水平随DN的进展而升高,C3aR在DN患者肾组织中的表达上调主要见于肾小管上皮细胞和肾小球足细胞;b.DN患者肾组织C3aR和C3a水平与患者肾组织损伤程度,特别是小管和小管间质损伤程度、肾小球足细胞损伤程度具有显著相关性;c.外加C3a激活C3aR可使高糖环境中的足细胞的细胞骨架发生明显改变、足细胞标记分子表达下调、足细胞通透性增加.这些结果说明:a.DN患者肾组织中确实存在C3a/C3aR轴过度活化的现象;b.C3a/C3aR轴的过度活化很可能在DN患者肾组织损伤,特别是小管和小管间质损伤、肾小球足细胞损伤中具有重要作用;c.可能通过破坏成熟足细胞特有的细胞骨架,改变足细胞标记分子表达,增加足细胞的通透性,C3a/C3aR轴过度活化参与DN足细胞损伤过程.本文不仅为C3a/C3aR通路参与DN病理过程提供了新的必不可少的临床证据,也增加了对C3a/C3aR通路过度活化致DN患者肾组织损伤机制,特别是肾小球足细胞损伤机制的了解,这对于拓展对DN病理机制的认识,发展DN防治新思路,无疑都是有益的.     

缺氧通过HIF-1α/NF-κB通路诱导肾小管上皮细胞C3aR表达

C3aR是补体C3a的受体．在肾脏,已发现C3aR表达于包括肾小管上皮细胞在内的多种细胞．在特定的病理情况下,C3aR表达上调并参与多种肾脏疾病的病理过程,但有关C3aR在肾脏细胞中的表达调控机制仍不清楚．小管间质缺氧是肾脏疾病中的一种常见现象,也是一种重要致病因素．为了探讨缺氧对C3aR的表达调控作用,本文利用体外缺氧模型,对模型条件下C3aR在肾小管上皮细胞中的表达变化情况进行了分析,同时检测了HIF-1α和NF-κB的表达变化及活化情况,以及HIF-1α和NF-κB抑制剂对C3aR的表达影响情况．结果发现缺氧可诱导C3aR mRNA及蛋白质水平的表达上调、HIF-1α和NF-κB的核转移．HIF-1α和NF-κB抑制剂可阻断缺氧对C3aR的诱导作用,且HIF-1α抑制剂可抑制NF-κB的核转移．这些结果说明缺氧可通过HIF-1α/NF-κB通路诱导肾小管上皮细胞C3aR的表达．考虑到C3aR活化可促进肾小管的损伤,　我们推测C3aR通路可能参与了缺氧和NF-κB诱导的肾小管损伤过程,可能是防治缺氧和NF-κB诱导肾组织损伤的一个新靶标．     

糖尿病肾病合并非糖尿病肾病的临床病理分析

目的：分析糖尿病肾病合并非糖尿病肾病的临床病理特点。方法：选取我院肾内科收治的临床诊断为糖尿病肾病的患者56 例,肾脏穿刺进行肾脏活体组织检查,通过病理诊断将患者分为两组,分别为糖尿病肾病组和糖尿病肾病合并非糖尿病肾病组, 比较两组患者的糖尿病病程、糖化血红蛋白、血压、血肌酐、血尿素氮、血尿酸、血清白蛋白、尿蛋白定量、血尿、视网膜病变。结果： 经肾脏组织活检,56例患者中NDRD 患者24 例(42.9%),DN患者32 例(57.1%);对24 例NDRD患者进行病理类型分类,其中IgA 肾病33.0%,膜性肾病25.0%、系膜增生性肾小球肾炎20.2%、高血压肾损害8.3%、微小病变4.2%、局灶节段硬化性肾炎4.2%、新 月体性肾小球肾炎4.2%。与DN组比较,NDRD 组糖尿病病程、糖化血红蛋白、血尿、视网膜病变均有差异(P＜0.05);而血肌酐、血 尿素氮、血尿酸、血清白蛋白、尿蛋白定量均无明显差异(P＞0.05)。结论：临床诊断的糖尿病肾病患者中有很大一部分实际上为糖 尿病肾病合并非糖尿病肾病,且以IgA 型肾病比较多见,糖尿病病程、糖化血红蛋白、血尿、视网膜病变对鉴别二者具有一定的指 导意义。     

大鼠肾组织PETN表达下调在糖尿病肾病发展中的作用

转化生长因子-β1(transforming growth factor-β1,TGF-β1)激活磷脂酰肌醇-3-激酶(phosphoinositide-3-kinase,PI3K)-蛋白激酶B(protein kinase B,PKB/Akt)通路与糖尿病肾病(diabetic nephropathy,DN)的发生发展密切相关,而第10号染色体缺失的磷酸酶和张力蛋白同源基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)可以负调节PI3K-PKB/Akt通路。本研究旨在观察糖尿病大鼠肾组织PTEN的表达变化及其在DN发生发展中的可能作用。16只Sprague-Dawley大鼠分成正常对照组和糖尿病组(n=8)。尾静脉注射链脲菌素(streptozotocin,STZ)复制糖尿病大鼠模型;12周处死大鼠,检测相应生化指标并计算肾脏指数;HE染色观察肾组织病理学改变;免疫组化和Western blotting检测PTEN、TGF-β1、PI3Kp110α、Akt1、p-Akt1(Ser473)、纤维连接蛋白(fibronectin,...     

周脂素在糖尿病肾病大鼠肾组织中的表达

目的观察大鼠糖尿病肾病(diabetic nephropathy,DN)模型的特点和尿LN对早期DN的诊断价值,探讨周脂素(perilipin,Plin)在DN大鼠肾脏中的表达情况。方法将14只SD雄性大鼠随机分为对照组(普通饲料)和糖尿病肾病模型组(高糖高脂饲料),对照组6只,模型组8只,饲养4周后模型组按照30mg/kg剂量注射1%链脲佐菌素(STZ),检测血糖≥16.7mmol/L,糖尿病模型制作成功,继续喂养6周,检测24h尿蛋白≥30mg/kg,糖尿病肾病模型制作成功。考马斯亮蓝检测24h尿蛋白、ELISA测尿层粘连蛋白,HE染色观察肾组织的病理变化,Real-time PCR及Western blot检测肾脏组织中perilipin表达情况。结果模型鼠24h尿蛋白≥30mg/kg,糖尿病肾病大鼠模型制作成功。和对照组大鼠相比,模型组的肾重/体重比明显增高(P<0.05),尿量、尿层粘连蛋白于5周出现升高、24h尿蛋白于6周时出现升高,且三项指标均随着时间不断增高。肾组织病理检查显示:肾小球肥大,基膜增生,微小血管瘤形成,肾小管管腔变形,上皮脱落、空泡样变,大量单核、淋巴等炎性细胞浸润,间质内胶原纤维增生。模型组大鼠肾组织Plin的mRNA及蛋白表达均明显的升高(P<0.05)。结论尿层粘连蛋白比24h尿蛋白升高得早,可作为早期糖尿病肾病的警示指标。Plin表达增高可能参与了糖尿病肾病肾病变过程,为进一步探讨糖尿病肾病的发病机制提供新的思路。     

葛根素对糖尿病大鼠肾功能及肾组织MMP-3、TIMP-1表达的影响

目的 探讨葛根素对糖尿病大鼠肾小球结构、功能及肾组织基质金属蛋白酶3(MMP-3)、组织抑制剂1(TIMP-1)表达的影响。方法 腹腔注射链脲佐菌素诱发大鼠糖尿病模型,每日ip葛根素注射液,共16周。采用原位杂交法检测肾小球TIMP-1 mRNA表达,流式细胞术和免疫组化检测肾皮质MMP-3、TIMP-1及Ⅳ型胶原、层粘连蛋白表达。结果 糖尿病组较对照组肾小球TIMP-l mRNA及蛋白表达增加,MMP-3、TIMP-1及Ⅳ型胶原、层粘连蛋白表达亦增加;葛根素用药组较糖尿病组TIMP-1 mRNA、蛋白及MMP-3、Ⅳ型胶原、层粘连蛋白表达减少。结论 葛根素对糖尿病大鼠肾功能、形态的影响具有保护作用,除降低血糖外,调节肾小球MMP-3、TIMP-1表达式从而减轻肾小球细胞外基质沉积也可能是其作用途径之一。     

人参皂甙Rg3对糖尿病肾病大鼠生化指标及病理的影响

目的:探讨人参皂苷Rg3对糖尿病肾病大鼠生化指标及病理改变的影响。方法:30只SD雄性大鼠按随机数字表法分为正常对照组、模型对照组和人参皂甙Rg3组。采用链脲佐菌素建立糖尿病肾病大鼠模型。造模成功后,Rg3治疗组每天以Rg3(0.5mg/kg)灌胃,余予以等量蒸馏水灌胃。30天后分别测3组大鼠血糖、24小时尿蛋白、血肌酐,并予以HE染色行肾组织活检。结果:与正常组比较,模型对照组大鼠血糖、24小时尿蛋白、血肌酐明显升高,肾小球体积增大,基底膜增厚、细膜基质增多,肾小球内炎细胞浸润(P0.01)。与模型对照组比较,人参皂甙Rg3组血糖、24小时尿蛋白、血肌酐明显降低,肾小球基底膜增厚程度减轻,细胞外基质堆积减少,差异具有显著性(P0.05)。结论:人参皂甙Rg3能显著降低糖尿病大鼠血糖、血肌酐、24 h尿蛋白,能改善其肾脏的病理损害。     

整合素相关激酶在糖尿病肾病的表达及其意义

目的探讨整合素相关激酶(Integrin-Linked Kinase,ILK)在糖尿病肾病患者肾组织中的表达及其意义.方法对3例正常肾组织,14例糖尿病肾病患者肾穿刺活检标本,应用免疫组织化学方法检测ILK和FN在肾组织的阳性表达强度,并作图像分析处理.结果在正常肾组织,ILK主要表达于肾小球脏层上皮细胞,系膜细胞和小管上皮细胞呈弱表达.在糖尿病肾病,ILK表达于肾小球脏层上皮细胞和系膜细胞,在萎缩变性的肾小管上皮细胞表达增强.在肾小球结节硬化时,ILK表达明显减少.此外,ILK和FN的表达量在糖尿病肾病早、中期成正相关(P<0.001),在糖尿病肾病晚期成负相关(P<0.05).结论 ILK在糖尿病肾病肾组织中表达量显著增加,并与FN的表达有一定的相关性,说明其可能通过促进细胞外基质FN等的积聚,在糖尿病肾小球硬化过程中发挥重要作用.     

孢素联合糖皮质激素对IgA肾病患者IgA、C3及IgA/C3影响研究

目的:探讨孢素联合糖皮质激素对IgA肾病患者IgA、C3及IgA/C3的影响。方法:我院收治的IgA肾病住院患者90例,按用药不同分为对照组与实验组。对照组予以醋酸泼尼松片口服,实验组在对照组基础上予以予环孢素软胶囊口服,治疗结束后对患者的血肌酐、血尿酸、24 h尿蛋白定量及IgA、C3、IgA/C3进行检测。结果:与对照组相比,实验组24 h尿蛋白定量水平较低,P0.05;IgA水平及IgA/C3水平较低,P0.05,差异具有统计学意义;两组患者的C3、血肌酐、血尿酸水平无显著差异,无统计学意义(P0.05)。结论:孢素联合糖皮质激素能够显著降低Ig A肾病患者Ig A水平及IgA/C3,对C3水平无影响,对临床有指导意义。     

Neutraligands of C5a can potentially occlude the interaction of C5a with the complement receptors C5aR1 and C5aR2

The complement system is central to the rapid immune response witnessed in vertebrates and invertebrates, which plays a crucial role in physiology and pathophysiology. Complement activation fuels the proteolytic cascade, which produces several complement fragments that interacts with a distinct set of complement receptors. Among all the complement fragments, C5a is one of the most potent anaphylatoxins, which exerts solid pro-inflammatory responses in a myriad of tissues by binding to the complement receptors such as C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2), which are part of the rhodopsin subfamily of G-protein coupled receptors. In terms of signaling cascade, recruitment of C5aR1 or C5aR2 by C5a triggers the association of either G-proteins or β-arrestins, providing a protective response under normal physiological conditions and a destructive response under pathophysiological conditions. As a result, both deficiency and unregulated activation of the complement lead to clinical conditions that require therapeutic intervention. Indeed, complement therapeutics targeting either the complement fragments or the complement receptors are being actively pursued by both industry and academia. In this context, the model structural complex of C5a–C5aR1 interactions, followed by a biophysical evaluation of the model complex, has been elaborated on earlier. In addition, through the drug repurposing strategy, we have shown that small molecule drugs such as raloxifene and prednisone may act as neutraligands of C5a by effectively binding to C5a and altering its biologically active molecular conformation. Very recently, structural models illustrating the intermolecular interaction of C5a with C5aR2 have also been elaborated by our group. In the current study, we provide the biophysical validation of the C5a-C5aR2 model complex by recruiting major synthetic peptide fragments of C5aR2 against C5a. In addition, the ability of the selected neutraligands to hinder the interaction of C5a with the peptide fragments derived from both C5aR1 and C5aR2 has also been explored. Overall, the computational and experimental data provided in the current study supports the idea that small molecule drugs targeting C5a can potentially neutralize C5a's ability to interact effectively with its cognate complement receptors, which can be beneficial in modulating the destructive signaling response of C5a under pathological conditions.     

Loss of C3a and C5a receptors promotes adipocyte browning and attenuates diet-induced obesity via activating inosine/A2aR pathway

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肾素-血管紧张素系统在糖尿病肾病肾脏足细胞损伤中的作用

糖尿病肾病(diabetic kidney disease,DKD)作为诱发终末期肾脏病(end-stage renal disease,ESRD)的主要原因,至今其病理机制仍不十分清楚.DKD病程中蛋白尿持续增多并伴随肾素-血管紧张素系统(renin-angiotensin system,RAS)过度激活.阻断RAS能改善蛋白尿,有良好的临床肾脏保护作用.足细胞表达RAS的各成员,作为肾小球滤过的最后屏障,其损伤与蛋白尿的发生关系密切.本文就RAS与足细胞损伤在DKD病理中作用作一简单综述.     

Dysregulation of microRNA-181b and TIMP3 is functionally involved in the pathogenesis of diabetic nephropathy

This study aimed to study the roleof microRNA (miR)-181b and its target TIMP3 in the development of diabetic nephropathy (DMN) via inhibiting the apoptosis of mesangial cells. Real-time polymerase chain reaction (RT-PCR) was adopted to compare the miR-181b expression between subjects with diabetic nephropathy (DN) and normal control. In addition, luciferase assays were utilized to explore the regulatory relationship between TIMP3 and miR-181b. Real-time PCR and densitometry analysis were conducted to measure the levels of TIMP3 mRNA/protein in DMN or in cells treated by miR-181b inhibitors, miR-181b mimics, and TIMP3 siRNA. And the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to study the effect of miR-181b on cell survival and apoptosis. miR-181b expression was much higher in the DN group, and the results of computational analysis identified TIMP3 as a miR-181b target. The luciferase activity of cells transfected with wild-type TIMP3 and mutant2 TIMP3 was significantly reduced, whereas the luciferase activity of cells transfected with mutant1 TIMP3 was evidently higher. Furthermore, a negative regulatory relationship was established between TIMP3 and miR-181b expression with a correlation efficient of −0.5351. The levels of TIMP3 mRNA/protein expression were apparently increased in the DN group. In addition, the treatment of cells with miR-181b mimics and TIMP3 siRNA remarkably lowered the levels of TIMP3 mRNA/protein, whereas the transfection of cells with miR-181b inhibitors notably elevated the expression of TIMP3 mRNA/protein. miR-181b promoted the survival of cells and inhibited their apoptosis. The miR-181b expression was related to the development of DMN and could be used as a prognosis biomarker of DMN in the patients with DM.     

MiR-92d-3p suppresses the progression of diabetic nephropathy renal fibrosis by inhibiting the C3/HMGB1/TGF-β1 pathway

The pathogenesis of diabetic nephropathy (DN) has not been fully elucidated. MicroRNAs (miRNAs) play an important role in the onset and development of DN renal fibrosis. Thus, the present study aimed to investigate the effect of miR-92d-3p on the progression of DN renal fibrosis. We used qRT-PCR to detect the expression levels of miR-92d-3p in the kidneys of patients with DN. Then, after transfecting lentiviruses containing miR-92d-3p into the kidneys of a DN mouse model and HK-2 cell line, we used qRT-PCR to detect the expression levels of miR-92d-3p, C3, HMGB1, TGF-β1, α-SMA, E-cadherin, and Col I. The expression levels of interleukin (IL) 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the HK-2 cells were detected through enzyme-linked immunosorbent assay (ELISA), and Western blotting and immunofluorescence were used in detecting the expression levels of fibronectin, α-SMA, E-cadherin, and vimentin. Results showed that the expression levels of miR-92d-3p in the kidney tissues of patients with DN and DN animal model mice decreased, and C3 stimulated HK-2 cells to produce inflammatory cytokines. The C3/HMGB1/TGF-β1 pathway was activated, and epithelial-to-interstitial transition (EMT) was induced in the HK-2 cells after human recombinant C3 and TGF-β1 protein were added. miR-92d-3p inhibited inflammatory factor production by C3 in the HK-2 cells and the activation of the C3/HMGB1/TGF-β1 pathway and EMT by C3 and TGF-β1. miR-92d-3p suppressed the progression of DN renal fibrosis by inhibiting the activation of the C3/HMGB1/TGF-β1 pathway and EMT.     

Chemerin/ChemR23 axis promotes inflammation of glomerular endothelial cells in diabetic nephropathy

Diabetic nephropathy (DN) is characterized by inflammation of renal tissue. Glomerular endothelial cells (GEnCs) play an important role in inflammation and protein leakage in urine in DN patients. Chemerin and its receptor ChemR23 are inducers of inflammation. The aim of this study was to investigate the function of chemerin/ChemR23 in GEnCs of DN patients. Immunohistochemical staining and qRT‐PCR were used to measure the expression of chemerin, ChemR23 and inflammatory factors in renal tissues of DN patients. Db/db mice were used as animal model. ChemR23 of DN mice was knocked down by injecting LV3‐shRNA into tail vein. Inflammation, physiological and pathological changes in each group was measured. GEnCs were cultured as an in vitro model to study potential signalling pathways. Results showed that expression of chemerin, ChemR23 and inflammatory factors increased in DN patients and mice. LV3‐shRNA alleviated renal damage and inflammation in DN mice. GEnCs stimulated by glucose showed increased chemerin, ChemR23 and inflammatory factors and decreased endothelial marker CD31. Both LV3‐shRNA and SB203580 (p38 MAPK inhibitor) attenuated chemerin‐induced inflammation and injury in GEnCs. Taken together, chemerin/ChemR23 axis played an important role in endothelial injury and inflammation in DN via the p38 MAPK signalling pathway. Suppression of ChemR23 alleviated DN damage.