Gangliosides in Podocyte Biology and Disease

Gangliosides constitute a subgroup of glycosphingolipids characterized by the presence of sialic acid residues in their structure. As constituents of cellular membranes, in particular of raft microdomains, they exert multiple functions, some of them capital in cell homeostasis. Their presence in cells is tightly regulated by a balanced expression and function of the enzymes responsible for their biosynthesis, ganglioside synthases, and their degradation, glycosidases. The dysregulation of their abundance results in rare and common diseases. In this review, we make a point on the relevance of gangliosides and some of their metabolic precursors, such as ceramides, in the function of podocytes, the main cellular component of the glomerular filtration barrier, as well as their implications in podocytopathies. The results presented in this review suggest the pertinence of clinical lipidomic studies targeting these metabolites.     

Inhibition of Toll-Like Receptor 4 Signaling Mitigates Microvascular Loss but Not Fibrosis in a Model of Ischemic Acute Kidney Injury

The development of chronic kidney disease (CKD) following an episode of acute kidney injury (AKI) is an increasingly recognized clinical problem. Inhibition of toll-like receptor 4 (TLR4) protects renal function in animal models of AKI and has become a viable therapeutic strategy in AKI. However, the impact of TLR4 inhibition on the chronic sequelae of AKI is unknown. Consequently, we examined the chronic effects of TLR4 inhibition in a model of ischemic AKI. Mice with a TLR4-deletion on a C57BL/6 background and wild-type (WT) background control mice (C57BL/6) were subjected to bilateral renal artery clamping for 19 min and reperfusion for up to 6 weeks. Despite the acute protective effect of TLR4 inhibition on renal function (serum creatinine 1.6 ± 0.4 mg/dL TLR4-deletion vs. 2.8 ± 0.3 mg/dL·WT) and rates of tubular apoptosis following ischemic AKI, we found no difference in neutrophil or macrophage infiltration. Furthermore, we observed significant protection from microvascular rarefaction at six weeks following injury with TLR4-deletion, but this did not alter development of fibrosis. In conclusion, we validate the acute protective effect of TLR4 signal inhibition in AKI but demonstrate that this protective effect does not mitigate the sequential fibrogenic response in this model of ischemic AKI.     

PTD-SARA融合蛋白的表达、纯化及鉴定

目的在大肠杆菌中表达PTD-SARA融合蛋白,并对其进行纯化和鉴定。方法使用大肠杆菌偏爱密码子合成PTD-SARA全长基因,将其克隆入载体pQE-30中,构建重组表达质粒pQE-30-PTD-SARA,转化感受态大肠杆菌M15,IPTG诱导表达。表达产物经Ni2+-NTA亲和层析纯化后,进行Western blot鉴定及N-端测序。结果重组表达质粒pQE-30-PTD-SARA经双酶切及测序鉴定证明构建正确。1.0mmol/LIPTG诱导4h,目的蛋白的表达量最高,约占菌体总蛋白的25%,主要以包涵体形式存在。纯化的融合蛋白纯度为94%,浓度为0.2mg/ml,且具有良好的反应原性,N-端有14个氨基酸。结论已成功表达并纯化了PTD-SARA融合蛋白,为其功能的研究奠定了基础,也为临床防治肾脏纤维化提供了一个新的策略。     

硫酸吲哚酚在慢性肾病并发症中的作用

慢性肾病（chronic kidney disease, CKD）是全球性公共卫生问题,其发生发展与体内尿毒症毒素暴露密切相关。硫酸吲哚酚（indoxyl sulfate, IS）是一种肠源性的蛋白结合型尿毒症毒素,由于CKD患者肾脏排泄功能被破坏,IS在CKD患者体内蓄积。IS不仅促进CKD进展,还诱导其他组织器官病变,进而引起CKD相关的并发症。本文综述了IS对CKD患者血管、肌肉、骨骼以及大脑的影响及其相应的机制,并总结通过清除IS治疗CKD的方法。     

细胞焦亡介导慢性间歇缺氧大鼠肾脏损伤及依达拉奉的干预作用

目的：探究依达拉奉对慢性间歇缺氧导致大鼠肾脏损伤的保护作用及其对Caspase-1介导的细胞焦亡信号通路的影响。方法：24只SPF级雄性SD大鼠随机分为正常对照（NC）组、间歇缺氧（IH）组、间歇缺氧+生理盐水（IH+NS）组、间歇缺氧+依达拉奉（IH+EDA）组,每组6只。将4组大鼠放置在密闭式饲养舱内造模,NC组舱内氧气浓度维持在21%左右,IH组、IH+NS组、IH+EDA组定时输入纯氧气、纯氮气、压缩空气,使舱内形成缺氧-复氧循环（60 s低氧期+60 s复氧期）,低氧期舱内氧浓度降至6%～7%,每日造模8 h（10∶00-18∶00）,同时IH+EDA组大鼠每天造模前按照5 mg/kg剂量标准予以腹腔注射依达拉奉,IH+NS组大鼠按照同等剂量标准腹腔注射生理盐水。造模8周后采集大鼠血液标本及肾脏组织标本,测定各组大鼠血肌酐（Crea）、尿素（Urea）水平;HE、Masson染色后光镜下观察肾脏病理形态变化、纤维化程度;化学法测定丙二醛（MDA）含量、超氧化物歧化酶（SOD）活力;免疫组织化学染色法测定肾组织NLRP3、Caspase-1、IL-1β蛋白表达水平;Western blot法测定肾组织Caspase-1、IL-1β蛋白表达水平;RT-PCR法测定消皮素D（gasdermin D, GSDMD）、IL-18 mRNA扩增水平。结果：间歇缺氧暴露后,大鼠血清Crea、Urea明显升高（P<0.01）,肾小管发生病理损害、肾单位球囊间隙出现胶原纤维沉积,MDA含量增加、SOD活力降低（P<0.01）,Caspase-1、NLRP3、IL-1β蛋白表达增加（P<0.01或P<0.05）、GSDMD mRNA、IL-18 mRNA扩增增加（P<0.01）;而经过依达拉奉干预后,上述指标呈现出与间歇低氧暴露后相反的变化趋势,肾脏病理损害减轻（P<0.01或P<0.05）。 结论：慢性间歇缺氧可能通过氧化应激活化Caspase-1参与的细胞焦亡信号通路介导肾脏损伤过程,而依达拉奉可能通过清除氧自由基、下调机体氧化应激水平,抑制焦亡通路的活化,起到保护肾脏的作用。     

Inhibition of extracellular signal-regulated kinases ameliorates hypertension-induced renal vascular remodeling in rat models

The aim of this study is to investigate the effect of the extracellular signal-regulated kinases 1/2 (ERK1/2) inhibitor, PD98059, on high blood pressure and related vascular changes. Blood pressure was recorded, thicknesses of renal small artery walls were measured and ERK1/2 immunoreactivity and erk2 mRNA in renal vascular smooth muscle cells (VSMCs) and endothelial cells were detected by immunohistochemistry and in situ hybridization in normotensive wistar kyoto (WKY) rats, spontaneously hypertensive rats (SHR) and PD98059-treated SHR. Compared with normo-tensive WKY rats, SHR developed hypertension at 8 weeks of age, thickened renal small artery wall and asymmetric arrangement of VSMCs at 16 and 24 weeks of age. Phospho-ERK1/2 immunoreactivity and erk2 mRNA expression levels were increased in VSMCs and endothelial cells of the renal small arteries in the SHR. Treating SHR with PD98059 reduced the spontaneous hypertension-induced vascular wall thickening. This effect was associated with suppressions of erk2 mRNA expression and ERK1/2 phosphorylation in VSMCs and endothelial cells of the renal small arteries. It is concluded that inhibition of ERK1/2 ameliorates hypertension induced vascular remodeling in renal small arteries.     

芸豆萌发处理各时期营养成分变化

通过对芸豆萌发前后各时期主要营养成分含量变化进行测定,分析其相关性,结果表明:蛋白质、脂肪、淀粉、还原糖、矿物元素、维生素C等含量与未萌发芸豆相比均发生显著变化(ρ<0.05)。蛋白质、游离氨基酸含量总体呈先上升后下降趋势,当芽长为2.01~2.50 cm时,蛋白质含量增加了20.62%。还原糖、维生素C含量总体呈上升趋势,当芽长大于3.51 cm时,分别增加了212.45%、243.66%。脂肪、淀粉含量总体呈下降趋势,当芽长大于3.51 cm时,分别下降了33.29%,19.12%。萌发后,营养成分发生改变,营养结构得以改善。     

芸豆蛋白酶解工艺的研究

以芸豆蛋白为原料研究芸豆抗氧化活性肽的酶解工艺。首先以DPPH.清除率和水解度为评价指标筛选最适用酶,并采用单因素及正交实验设计优化酶解工艺。结果表明,Alcalase碱性蛋白酶对芸豆蛋白水解能力最强,酶解产物的抗氧化能力最高;最佳酶解工艺条件为底物浓度4.0%,温度55℃,加酶量2000u.g-1,pH9.0,时间6h,该条件下水解度和DPPH.清除率分别为16.16%和74.10%,比优化前分别提高4.95%和7.63%;芸豆活性肽显示出较强的抗氧化活性。     

酰化对芸豆分离蛋白热学特性的影响研究

从参与酰化反应基团的视角,运用热分析(DSC)技术,研究了不同酰化阶段芸豆分离蛋白热学性质的变化规律。芸豆分离蛋白(KPI)的酰化过程存在两个主要的酰化阶段,酸酐-蛋白比为0～0.1(乙酰化)和0～0.2(琥珀酰化)g/g,为ε-氨基(Lys)酰化阶段(N-酰化);再增加酸酐与蛋白比,ε-氨基酰化基本完成,反应进入羟基(Thr,Ser)酰化阶段(O-酰化)。在N-酰化阶段,琥珀酰化诱导KPI的热稳定性增加,而焓变(ΔH)略有降低,乙酰化不影响KPI的热稳定性和焓变(ΔH);羟基酰化阶段,酰化导致KPI变性温度(Td)降低,伴随焓变(ΔH)急剧下降。     

牛磺酸对顺铂引起的小鼠肾髓质ATP酶活性变化的影响

目的 :探讨牛磺酸对顺铂引起的小鼠肾髓质ATP酶活性的影响 ;方法 :4 0只小鼠随机分为正常对照组、CDDP组、牛磺酸 2 0 0mg组 (Tau2 0 0 )和牛磺酸 4 0 0mg组 (Tau4 0 0 ) ,分取肾髓质、制备匀浆 ,用生化法测定Na ,K -ATP酶、Ca2 -ATP酶和Mg2 -ATP酶活性 ;结果 :CDDP组、Tau2 0 0组、Tau4 0 0组肾髓质Na ,K -ATP酶、Ca2 -ATP酶和Mg2 -ATP酶活性均明显低于正常对照组 (P <0 .0 1)。Tau2 0 0组、Tau4 0 0组肾髓质Na ,K -ATP酶和Ca2 -ATP酶活性均明显高于CDDP组 (P <0 .0 1或P <0 .0 5 ) ,而Mg2 -ATP酶活性与CDDP组相比无显著差别 (P >0 .0 5 ) ;结论 :牛磺酸可明显增加肾髓质Na ,K -ATP酶和Ca2 -ATP酶活性 ,对顺铂引起的肾毒性具有防治作用。