糖基化终末产物对大鼠肾小球系膜细胞尾加压素Ⅱ及G蛋白偶联受体mRNA表达的影响

目的观察不同浓度糖基化终末产物(Advanced glycation end products,AGEs)及AGEs作用不同时间对大鼠肾小球系膜细胞尾加压素Ⅱ(UrotensinⅡ,UⅡ)及G蛋白偶联受体(G-protein-couple receptor,GPR14)mRNA表达的影响。方法制备AGE-BSA,体外培养大鼠肾小球系膜细胞(Mesangial Cells,MC),加入不同浓度的AGE-BSA(终浓度分别为0、25、50、100和200mg/L),37℃孵育24h;加入100mg/L AGE-BSA,分别培养0、2、8、16和24h,以不含葡萄糖的BSA作为对照。收集细胞,采用RT-PCR检测各组MC UⅡ及GPR14mRNA的表达。结果 AGE-BSA各组MC UⅡ及GPR14mRNA的表达量均随AGEs浓度的增加而增加,50、100和200mg/L与0mg/L组比较,差异有统计学意义(P<0.05);100mg/L AGE-BSA各组MC UⅡ及GPR14mRNA的表达量随着作用时间的延长而增加,作用8、16、24h组与0h组比较,差异有统计学意义(P<0.05)。BSA组MC UⅡ及GPR14mRNA的表达量无明显增加(P>0.05)。结论 AGEs能上调大鼠MC UⅡ及GPR14mRNA的表达。     

慢性肾小管间质病变的中医药研究概况

近年来,多项研究表明,各种肾脏病进行性肾功能恶化主要取决于肾小管间质损伤的严重程度,因此,肾小管间质病变(TIL)受到重视.TIL发病机理十分复杂,早期表现可为肾间质局部炎症,随后出现细胞因子分泌,继而肾小管上皮细胞等表型转化、细胞凋亡或细胞过度增殖,细胞外基质(ECM)合成增多、降解减少,造成ECM过度沉积,最终导致肾间质纤维化(RIF).现将中医中药对TIL的研究进展概述如下.     

大孔球形纤维素阴离子交换树脂PSC-AN对蛋白质的吸附性能研究CSCD

以自制的大孔球形纤维素阴离子交换树脂PSC-AN为吸附剂,以牛血清白蛋白(BSA)为目标蛋白,考察了该阴离子交换树脂对BSA的吸附性能,发现其对BSA的饱和吸附量为204.4 mg/m L,吸附行为满足Freundlich方程,建立了大孔球形纤维素吸附树脂PSC-AN吸附测定水中牛血清白蛋白含量的方法。     

Cu(Ⅱ)-SCN~-二元配合物与蛋白质的相互作用的电化学研究

用电化学方法对Cu(Ⅱ)-SCN-二元配合物与牛血清白蛋白(BSA)的相互作用进行了研究,发现在0.04 mol/L的高氯酸介质中,Cu(Ⅱ)-SCN-二元配合物能与牛血清白蛋白生成一种非电活性的复合物,导致Cu(Ⅱ)-SCN-二元配合物的还原峰电流降低,峰电流降低值Δip如在一定范围内与BSA的浓度成线性关系,线性范围为0.5~5.5 mg/L,相关系数为0.994,检测限为0.5mg/L,所以该法可用于蛋白质含量的测定。     

紫外和圆二色光谱研究三环唑与牛血清白蛋白的相互作用

通过紫外(UV)和圆二色(CD)光谱,研究三环唑与牛血清白蛋白(BSA)的相互作用及其浓度变化对牛血清白蛋白(BSA)的影响.研究表明:三环唑对牛血清白蛋白的影响是多方面的.紫外吸收光谱表明三环唑与牛血清白蛋白(BSA)借助分子间力形成超分子基态复合物,此外由于它们之间的疏水作用,导致BSA紫外吸收强度随三环唑浓度增大逐渐增强;CD光谱表明三环唑与BSA间的疏水作用使BSA的肽链发生收缩、重排.三环唑导致BSA的构象发生变化.     

氟伐他汀对糖尿病大鼠肾脏CTGF表达的影响

目的观察氟伐他汀对糖尿病大鼠肾脏结缔组织生长因子(CTGF)表达的影响。方法用链脲佐菌素复制糖尿病大鼠模型,成模后给予氟伐他汀治疗。采用ELISA法检测尿微量白蛋白浓度;Western blot和RT-PCR法检测大鼠肾皮质CT-GF蛋白及mRNA的表达水平;免疫组化法检测肾小球内CTGF和FN的表达。结果氟伐他汀治疗组大鼠尿白蛋白排泄率、肾脏肥大指数(KHI)、肾皮质中CTGF的mRNA及蛋白表达、肾小球内CTGF和FN的表达较糖尿病组大鼠均明显降低。结论氟伐他汀能降低肾小球内CTGF及FN的表达,减轻肾小球肥大,起到抑制和延缓糖尿病肾病进展的作用。     

热变性对蛋白质结构及泡沫行为的影响

以牛血清白蛋白(BSA)和鸡蛋白蛋白(albumin)为模型蛋白,研究了热变性对蛋白质起泡性能和分子结构的影响. 结果表明,热变性后的BSA起泡能力下降,泡沫稳定性有增强的趋势. 热变性使BSA分子表面巯基含量下降,分子之间发生缔合,表面疏水性下降. 而albumin在热变性后的起泡能力及泡沫稳定性都大大提高,热变性使albumin分子展开,表面巯基含量增加. 实验证明,蛋白质分子的表面疏水性是决定其起泡能力的重要因素之一,蛋白质分子之间的相互作用对泡沫的稳定性有很大的影响.     

重组蛋白转导结构域-SARA融合蛋白生物学功能的初步研究

目的初步研究重组蛋白转导结构域(Protein transduction domain,PTD)-SARA融合蛋白(PTD-SARA)抗肾脏纤维化的生物学功能。方法采用免疫细胞化学法检测重组PTD-SARA融合蛋白的穿膜作用;通过显微镜观察人肾小管上皮细胞HK2形态学变化,Western blot法测定上皮细胞钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)和磷酸化Smad3蛋白水平,以比较PTD-SARA融合蛋白和SARA蛋白抑制转化生长因子-β1(TGF-β1)诱发的肾小管上皮细胞向间质细胞转分化作用。结果重组PTD-SARA融合蛋白可高效地转入细胞胞质与胞核;PTD-SARA融合蛋白与SARA蛋白相比,能更显著地上调E-cadherin蛋白的表达,下调α-SMA和磷酸化Smad3蛋白的表达,且差异有统计学意义(P<0.05)。结论与SARA蛋白相比,PTD-SARA融合蛋白可以高效地进入细胞胞质及胞核,并能明显地抑制TGF-β1诱发的肾小管上皮细胞向间质细胞转分化作用,为进一步研究重组PTD-SARA融合蛋白防治肾脏纤维化奠定了基础。     

应用酶免疫方法检测生物制品中残余牛血清白蛋白含量

目的应用酶免疫方法检测生物制品中残余牛血清白蛋白(BSA)含量。方法制备高亲和力、高纯度的抗BSA单克隆抗体作为包被抗体,高纯度、高效价的兔抗BSA多克隆抗体为酶标抗体,采用ELISA夹心方法,以系列含量的BSA溶液作为标准,制备标准曲线,对样品中所含BSA进行定量。结果优化了酶免疫反应条件,标准曲线线性范围内r≥0.98,经验证,该方法对BSA的最低检测限为2.5ng/ml。分别检测5、10、30 ng/ml含量的BSA时,试验内(n=11)和试验间(n=3)测定的变异系数分别为2.3％-4.9％和4.0％-6.1％;回收率均值分别为104％、123％和109％。未见该方法与人、马、猪血清白蛋白之间的交叉反应。结论该法敏感度高,准确性、重复性和稳定性好,可用于生物制品的质量控制。     

锌(Ⅱ) - 2,2''-联吡啶络合吸附波法测定血清白蛋白

在pH值6.3的HAc-NaAc介质中,血清白蛋白使锌(Ⅱ) - 2,2′-联吡啶络合物在-1.20 V(vs.SCE)处的络合吸附波还原峰电流降低,峰电流降低值与加入的牛血清白蛋白(BSA)或人血清白蛋白(HSA)的浓度在一定范围内呈线性关系.BSA和HSA的线性范围分别为0.5～40.0 mg·L-1、0.5～50.0 mg·L-1,检出限均为0.2 mg·L-1.应用该法测定了人血清样品中总蛋白含量,结果令人满意.     

尾加压素Ⅱ对大鼠近端肾小管上皮NRK-52E细胞增殖和细胞周期的影响

目的探讨尾加压素Ⅱ(UⅡ)对大鼠近端肾小管上皮NRK-52E细胞增殖和细胞周期的影响及其机制。方法在NRK-52E细胞培养液中加入10-10、10-9、10-8和10-7mol/L UⅡ,同时设UⅡ活性阻断组:UⅡ+尼卡地平和UⅡ+EDTA,以单纯DMEM为对照组。培养48 h后,采用5-溴-2′-脱氧尿嘧啶核苷(BrdU)掺入法检测各组细胞的增殖活性,流式细胞术分析细胞周期。结果UⅡ(10-10~10-8mol/L)以浓度依赖方式促进NRK-52E细胞BrdU掺入(A值分别为0.491±0.038、0.291±0.024和0.281±0.037),其中以10-8mol/L UⅡ的作用最明显,10-7mol/L UⅡ对NRK-52E细胞增殖的影响与对照组比较差异无显著意义。UⅡ影响NRK-52E细胞周期,在10-10~10-8mol/L浓度范围内增加S期细胞百分比(分别为26.96%±3.35%、44.26%±3.28%、48.12%±2.22%)。尼卡地平和EDTA均可以降低UⅡ诱导NRK-52E细胞的BrdU掺入增加,降低S期细胞百分比。结论UⅡ具有较强促进NRK-52E细胞增殖的作用,但大剂量则无此作用,其诱导NRK-52E细胞增殖作用部分是通过Ca2+内流来介导的。     

The Protective Effect of Glycyrrhizic Acid on Renal Tubular Epithelial Cell Injury Induced by High Glucose

The aim of this study was to determine the beneficial effect of glycyrrhizic acid (GA) on type 2 diabetic nephropathy using renal tubular epithelial cell line (NRK-52E). The cells are divided into normal group (NG), high glucose group (HG), and treatment group (HG + GA). The methylthiazoletetrazolium (MTT) assay was used to detect the cell proliferation. Cell cycle analysis was performed using flow cytometry. Model driven architecture (MDA), reactive oxygen species (ROS) and superoxide dismutase (SOD) were also measured. Electron microscopy and histological were used to detect the changes in cell ultrastructure. The phosphorylation of AMP-activated protein kinase (AMPK), silent information regulator T1 (SIRT1), manganese-superoxide dismutase (Mn-SOD) and transforming growth factor-β1 (TGF-β1) were assessed by immunohistochemistry, immunofluorescence, and western blotting. Real-time fluorescent quantitative PCR (RT-qPCR) was used to measure Mn-SOD and PPARγ co-activator 1α (PGC-1a) mRNA. We find that high glucose increases NRK-52E cell proliferation and TGF-β1 expression, but decreases expression of AMPK, SIRT1 and Mn-SOD. These effects are significantly attenuated by GA. Our findings suggest that GA has protective effects against high glucose-induced cell proliferation and oxidative stress at least in part by increasing AMPK, SIRT1 and Mn-SOD expression in NRK-52E cells.     

Necrostatin-1 Attenuates Ischemia Injury Induced Cell Death in Rat Tubular Cell Line NRK-52E through Decreased Drp1 Expression

Necrostatin-1 (Nec-1) inhibits necroptosis and is usually regarded as having no effect on other cell deaths. Here, this study explored whether the addition of Nec-1 has an effect on cell death induced by simulated ischemia injury in rat tubular cell line NRK-52E. In addition, we also investigated the mechanism of Nec-1 attenuates cell death in this renal ischemia model. The NRK-52E cells were incubated with TNF-α + antimycinA (TA) for 24 h with or without Nec-1. Cell death was observed under fluorescent microscope and quantified by flow cytometry. Cell viabilities were detected by MTT assay. The protein expression of dynamin-related protein 1 (Drp1) was detected by Western blotting and immunofluorescence assay. Increased cell death in simulated ischemia injury of NRK-52E cells were markedly attenuated in the Nec-1 pretreated ischemia injury group. Meanwhile, cell viability was significantly improved after using Nec-1. In addition, we also observed that the protein expression of Drp1, a mediator of mitochondrial fission, was significantly increased in simulated ischemia injury group. Increased Drp1 expression in the ischemia injury group can be abolished by Nec-1 or Drp1-knock down, accompanied with decreased cell death and improved cell viabilities. These results suggest that Nec-1 may inhibit cell death induced by simulated ischemia injury in the rat tubular cell line NRK-52E through decreased Drp1 expression.     

Advanced Glycation End Product-Induced Astrocytic Differentiation of Cultured Neurospheres through Inhibition of Notch-Hes1 Pathway-Mediated Neurogenesis

This study aims to investigate the roles of the Notch-Hes1 pathway in the advanced glycation end product (AGE)-mediated differentiation of neural stem cells (NSCs). We prepared pLentiLox3.7 lentiviral vectors that express short hairpin RNA (shRNA) against Notch1 and transfected it into NSCs. Cell differentiation was analyzed under confocal laser-scanning microscopy. The percentage of neurons and astrocytes was quantified by normalizing the total number of TUJ1⁺ (Neuron-specific class III β-tubulin) and GFAP⁺ (Glial fibrillary acidic protein) cells to the total number of Hoechst 33342-labeled cell nuclei. The protein and gene expression of Notch-Hes1 pathway components was examined via western blot analysis and real-time PCR. After 1 week of incubation, we found that AGE-bovine serum albumin (BSA) (400 μg/mL) induced the astrocytic differentiation of cultured neurospheres and inhibited neuronal formation. The expression of Notch-Hes1 pathway components was upregulated in the cells in the AGE-BSA culture medium. Immunoblot analysis indicated that shRNA silencing of Notch1 expression in NSCs significantly increases neurogenesis and suppresses astrocytic differentiation in NSCs incubated with AGE-BSA. AGEs promote the astrocytic differentiation of cultured neurospheres by inhibiting neurogenesis through the Notch-Hes1 pathway, providing a potential therapeutic target for hyperglycemia-related cognitive deficits.     

DNase I Induces Other Endonucleases in Kidney Tubular Epithelial Cells by Its DNA-Degrading Activity

Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.     

AGEs-Induced IL-6 Synthesis Precedes RAGE Up-Regulation in HEK 293 Cells: An Alternative Inflammatory Mechanism?

Advanced glycation end products (AGEs) can activate the inflammatory pathways involved in diabetic nephropathy. Understanding these molecular pathways could contribute to therapeutic strategies for diabetes complications. We evaluated the modulation of inflammatory and oxidative markers, as well as the protective mechanisms employed by human embryonic kidney cells (HEK 293) upon exposure to 200 μg/mL bovine serum albumine (BSA) or AGEs–BSA for 12, 24 and 48 h. The mRNA and protein expression levels of AGEs receptor (RAGE) and heat shock proteins (HSPs) 27, 60 and 70, the activity of antioxidant enzymes and the expression levels of eight cytokines were analysed. Cell damage via oxidative mechanisms was evaluated by glutathione and malondialdehyde levels. The data revealed two different time scale responses. First, the up-regulation of interleukin-6 (IL-6), HSP 27 and high catalase activity were detected as early as 12 h after exposure to AGEs–BSA, while the second response, after 24 h, consisted of NF-κB p65, RAGE, HSP 70 and inflammatory cytokine up-regulation, glutathione depletion, malondialdehyde increase and the activation of antioxidant enzymes. IL-6 might be important in the early ignition of inflammatory responses, while the cellular redox imbalance, RAGE activation and NF-κB p65 increased expression further enhance inflammatory signals in HEK 293 cells.     

Danegaptide Prevents TGFβ1-Induced Damage in Human Proximal Tubule Epithelial Cells of the Kidney

Chronic kidney disease (CKD) is a global health problem associated with a number of comorbidities. Recent evidence implicates increased hemichannel-mediated release of adenosine triphosphate (ATP) in the progression of tubulointerstitial fibrosis, the main underlying pathology of CKD. Here, we evaluate the effect of danegaptide on blocking hemichannel-mediated changes in the expression and function of proteins associated with disease progression in tubular epithelial kidney cells. Primary human proximal tubule epithelial cells (hPTECs) were treated with the beta1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± danegaptide. qRT-PCR and immunoblotting confirmed mRNA and protein expression, whilst a cytokine antibody array assessed the expression/secretion of proinflammatory and profibrotic cytokines. Carboxyfluorescein dye uptake and ATP biosensing measured hemichannel activity and ATP release, whilst transepithelial electrical resistance was used to assess paracellular permeability. Danegaptide negated carboxyfluorescein dye uptake and ATP release and protected against protein changes associated with tubular injury. Blocking Cx43-mediated ATP release was paralleled by partial restoration of the expression of cell cycle inhibitors, adherens and tight junction proteins and decreased paracellular permeability. Furthermore, danegaptide inhibited TGFβ1-induced changes in the expression and secretion of key adipokines, cytokines, chemokines, growth factors and interleukins. The data suggest that as a gap junction modulator and hemichannel blocker, danegaptide has potential in the future treatment of CKD.     

Simultaneous Use of ROCK Inhibitors and EP2 Agonists Induces Unexpected Effects on Adipogenesis and the Physical Properties of 3T3-L1 Preadipocytes

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.     

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1-Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention.