PTEN在糖尿病肾病患者足细胞损伤中的作用

目的：探讨磷酸酰肌醇3激酶/蛋白激酶B( phosphoinositide 3 kinese/protein kinase B, PI3K/Akt)信号通路抑制因子PTEN与糖尿病肾病( diabetic nephropathy, DN)患者足细胞损伤的关系。方法收集30例DN患者和10例健康志愿者的24 h尿标本,采用酶联免疫吸附实验( enzyme linked immunosorbent assay, ELISA)检测患者尿液中足盂蛋白( podocalyxin, PCX)的水平;肾活检组织进行形态学观察,根据肾小球病变将DN患者分为3组,免疫组化法检测各组肾小球内p-Akt和PTEN的表达。结果 DN患者尿液中PCX的水平明显高于健康对照组,并随肾小球病变加重其水平逐渐上升;p-Akt和PTEN在DN患者肾小球的表达有所上调,但随着肾小球病变的加重其表达逐渐减少;DN患者尿液中PCX水平与PTEN表达呈负相关,与24 h尿蛋白呈正相关。结论 PTEN表达下调可能通过改变Akt的活化状态,从而在DN患者足细胞损伤中发挥一定作用。     

膜性肾病肾活检组织中TRPC6和podocalyxin表达改变及其意义

目的检测膜性肾病(membranous nephropathy,MN)患者肾活检组织中肾小球足细胞瞬时受体电位阳离子通道蛋白6(transient receptor potential cation channel 6,TRPC6)和podocalyxin的表达和分布,探讨足细胞蛋白TRPC6和podocalyxin在MN蛋白尿发生中的作用。方法采用常规组织病理以及免疫组化检查,并行免疫荧光双套色染色于激光共聚焦显微镜下观察TRPC6、podocalyxin在各组肾组织中表达及分布的改变,以计算机图像分析系统进行半定量分析。结果对照组肾组织podocalyxin沿肾小球毛细血管壁连续均匀分布,阳性荧光信号表达强,MN组表达减弱,且分布不均或节段性缺失,部分呈点状、短线状不连续分布,肾病综合征组较非肾病综合征组减弱更明显;TRPC6在对照组肾小球有一定的表达,沿肾小球基膜呈均匀连续线型分布,MN组TRPC6荧光强度有不同程度增强,且呈点状、团块状不均匀分布,肾病综合征组较非肾病综合征组更明显。结论 TRPC6和podocalyxin在正常肾组织沿肾小球基膜呈连续线状均匀分布;MN患者肾小球内TRPC6表达增加,podocalyxin表达减少,且分布形式亦发生变化,提示TRPC6和podocalyxin等足细胞相关蛋白表达改变及分布异常可能是MN蛋白尿发生的重要病理机制。     

Sos2抑制NFATc1介导的糖尿病肾病足细胞损伤的机制研究

目的:观察高糖刺激对体外培养的小鼠肾足细胞鸟苷酸交换因子Sos2(Son of Sevenless homolog2)表达的影响,并初步探讨Sos2在高糖诱导足细胞损伤中的作用及其可能的分子机制。方法:通过免疫荧光染色及激光共聚焦显微镜观察Sos2在糖尿病肾病患者足细胞中的表达;体外培养小鼠永生化足细胞,以高糖(30 mmol/L葡萄糖)刺激足细胞48 h,采用RT-PCR、Western blot和免疫荧光检测高糖刺激下足细胞Sos2的mRNA及蛋白表达;采用Western bolt实验、免疫荧光及划痕实验检测Sos2过表达及沉默后podocin的表达、足细胞的活动性及NFATc1的入核情况;并采用RT-PCR检测NFATc1下游目的基因转录情况。结果:Sos2在糖尿病肾病患者的足细胞及高糖刺激体外培养的足细胞中表达显著降低(P 0. 05);沉默Sos2后,足细胞标志蛋白podocin表达显著降低,足细胞活动性增加,NFATc1入核增加,NFATc1下游目的基因转录增加(P 0. 05);与之相反,过表达Sos2组podocin表达显著增高,足细胞的活动性降低,NFATc1的入核减少,NFATc1下游目的基因转录降低(P 0. 05)。结论:Sos2可能通过抑制NFATc1入核而减轻糖尿病肾病足细胞损伤。     

血、尿IL-6变化在糖尿病肾病中的意义

目的 探讨血、尿IL-6变化在糖尿病肾病诊断治疗中的意义.方法 将实验对象分为观察组和对照组,观察组52人,对照组40人.根据尿白蛋白排泄率(UAER)将观察组又分为两个小组:分别为微量蛋白尿组DN1(24例)和临床蛋白尿组DN2(28例).对照组选取40名健康成年人.观察各组血、尿IL-6水平.结果 ①观察组病人血、...     

厄贝沙坦对早期糖尿病肾病大鼠足细胞损伤及整合素连接激酶的影响

目的: 研究血管紧张素Ⅱ受体拮抗剂厄贝沙坦(Irb)对早期糖尿病肾病大鼠足细胞损伤及整合素连接激酶(ILK)的影响。方法: 将注射链脲佐菌素(STZ)的自发性高血压大鼠(SHR)分为糖尿病肾病组(DN,n=8)和糖尿病厄贝沙坦治疗组(DN+Irb,n=9),与SHR遗传背景相同周龄和体重相当的非高血压Wistar-Kyoto大鼠作为正常对照(control,n=11)。观察生化指标变化和病理改变情况,以及Irb对糖尿病肾病大鼠足细胞损伤和ILK的影响。结果: 与对照组相比,DN组大鼠表现为高血糖、高血压、高血脂、胰岛素抵抗和蛋白尿;病理上出现系膜基质增生,足细胞数目减少和足细胞损伤;ILK mRNA和蛋白表达水平明显上调。Irb治疗显著降低血压和蛋白尿,抑制足细胞数目减少,减轻足细胞损伤,下调ILK的表达。结论: 糖尿病大鼠早期肾脏足细胞损伤和ILK表达增加,Irb可能通过下调ILK表达而发挥足细胞保护作用。     

小檗碱对糖尿病肾病大鼠C反应蛋白的影响

目的:观察小檗碱对糖尿病肾病大鼠C反应蛋白的影响。方法:将48只雄性SD大鼠随机分为空白对照组、模型组、小檗碱75、150及300mg.kg-1组、罗格列酮组六组,每组8只。除正常对照组外,其余各组大鼠腹腔注射链脲佐菌素(STZ,55mg.kg-1),成功造模后小檗碱各组及罗格列酮组分别灌胃给予75、150、300mg.kg-1小檗碱以及4mg.kg-1罗格列酮,连续6w后检测大鼠血糖、体重,HE染色检测肾脏病理改变,ELISA法检测C反应蛋白活性。结果:与正常对照组相比,糖尿病组血糖与C反应蛋白活性明显升高(P<0.05),与模型组比较,小檗碱各组血糖与C反应蛋白活性明显降低(P<0.05),有统计学意义。结论:小檗碱可降低糖尿病大鼠血糖和C反应蛋白,减轻糖尿病肾病。     

Risk genotypes and haplotypes of the GLUT1 gene for type 2 diabetic nephropathy in the Tunisian population

Objective: Diabetic nephropathy (DN) is a long-term complication of both type 1 and type 2 diabetes. Genetic studies on DN have been of little help so far, since several genetic association studies have shown conflicting results. Here we report the findings of a case-control study on five SNPs in the glucose transporter 1 (GLUT1) gene. The study investigated the association of five GLUT1 genotypes and haplotypes with DN.

Research design and methods: All subjects, 126 DN (cases) and 273 type 2 diabetes (controls), were genotyped using the polymerase chain reaction restriction fragment length polymorphism.

Results: The TT and the AA genotypes of the Haell and Enh2 SNP1, increased the risk of DN. The study also identified CGT as the highest risk haplotype (4.4-fold) followed by CAT with an increased risk of DN of 2.6-fold.

Conclusions: The GLUT1 gene confers susceptibility to DN in type 2 diabetes patients in the Tunisian population.     

The polymorphism of manganese superoxide dismutase is associated with diabetic nephropathy in Japanese type 2 diabetic patients

 We evaluated the relationship of an alanine or valine polymorphism at amino acid sequence 16 [Val(16)Ala] of manganese superoxide dismutase (Mn-SOD) with diabetes and diabetic nephropathy in Japanese type 2 diabetic patients. Val(16)Ala genotyping of Mn-SOD was done by polymerase chain reaction-restriction fragment length polymorphism with a restriction enzyme (Bsaw I) in 478 Japanese type 2 diabetic patients and 261 nondiabetic Japanese healthy subjects. The genotype distribution of diabetic and nondiabetic subjects was then compared, and the association of genotype with diabetic nephropathy was evaluated in the diabetic patients. The allele frequency and genotype of the diabetic patients were not different from those of the healthy nondiabetic subjects. The VV type showed a significantly higher frequency in the diabetic patients with nephropathy than did the AA or VA type [VV type: normoalbuminuria 70.8%, microalbuminuria 84.8% (P = 0.0057), macroalbuminuria 84.1% (P = 0.0128)]. Furthermore, logistic regression analysis showed that this polymorphism is associated with diabetic nephropathy independently (odds ratio = 0.461925, P = 0.03). Accordingly, the Val(16)Ala polymorphism of Mn-SOD may be unrelated to the etiology of type 2 diabetes, but it seems to be associated with diabetic nephropathy in Japanese type 2 diabetic patients. Received: August 5, 2002 / Accepted: December 3, 2002 Correspondence to:Y. Tanaka     

血管紧张素原基因M235T分子变异与2型糖尿病肾病的关系

目的　探讨血管紧张素原（ａｎｇｉｏｔｅｎｓｉｎｏｇｅｎ , Ａ Ｇ Ｔ） 基因 Ｍ２３５ Ｔ 分子变异与中国人无肾病并发症的２ 型糖尿病（ｄｉａｂｅｔｅｓ ｍ ｅｌｌｉｔｕｓ , Ｄ Ｍ） 、２ 型糖尿病肾病（ｄｉａｂｅｔｉｃ ｎｅｐｈｒｏｐａｔｈｙ , Ｄ Ｎ） 的关系。方法　用 Ｐ Ｃ Ｒ及 Ｒ Ｆ Ｌ Ｐ 方法对８４ 例 Ｄ Ｍ、９６ 例 Ｄ Ｎ 及９８ 名正常对照进行了 Ａ Ｇ Ｔ 基因 Ｍ２３５ Ｔ 多态性的检测。结果　 Ｄ Ｎ 组 Ｔ 等位基因频率０８２ , Ｔ Ｔ 基因型频率０７０ ,与对照组（０６３ ,０４３） 比较有显著差异（ Ｐ＝ ０００３ , Ｐ＝００００４） ;校正了 Ｄ Ｎ 的几种危险因素后, Ｔ Ｔ 基因型对 Ｄ Ｎ 的 Ｏ Ｒ 为３４７（９５ ％ Ｃ Ｉ 为１５１ ～７９４ , Ｐ＝０００３３） 。 Ｄ Ｍ 组基因型频率分布与对照组比较无显著差异（ Ｐ＞ ００５） 。结论　 Ａ Ｇ Ｔ 基因 Ｔ Ｔ 型可能是中国人群２ 型糖尿病肾病的独立危险因素之一。     

2型糖尿病及其肾病与血管生成素2基因多态性的关联分析

目的 研究2型糖尿病(type 2 diabetes mellitus,T2DM)及其肾病(diabetic nephropathy,DN)发生、发展与血管生成素2(angiopoietin-2,Ang-2)基因多态性的关联性.方法 应用等位基因特异聚合酶链反应检验无亲缘关系汉族人群的2型糖尿病及其肾病患者221例、正常对照104名Ang-2基因单核苷酸多态性(single nucleotide polymorphism,SNP)759T/G、1078A/G、1233A/G在病例组中的频率,并用尿白蛋白排泄率、炎症标志物单核细胞趋化蛋白浓度变化探讨上述SNP与T2DM、DN发生发展的关联.结果 (1) Ang-2 759 T/G、1078 A/G基因型频率和等位基因频率在各组中的差异无统计学意义,而1233 A/G基因型频率和等位基因频率的差异有统计学意义(P＜0.05);(2)在校正年龄和性别等因素后,1233A/GSNP G等位基因患T2DM和DN的风险分别是A等位基因的2.265倍(95％ CI:1.223～1.402,P=0.031)和1.789倍(95％ CI:0.889～1.021,P=0.012);(3) Ang-2 1233A/G G等位基因与DN发生相关(r=1.321,OR=1.427,95％ CI:2.324～4.177,P=0.034).结论 Ang-2 1233A/G SNP与2型糖尿病的发生有关;AG+ GG基因型和等位基因G可能是DN发生、发展的风险因素.     

In situ localization of type III and type IV collagen-expressing cells in human diabetic nephropathy

Nodular intercapillary glomerulosclerosis is the most typical lesion of diabetic nephropathy (DN) and is characterized by increased extracellular matrix (ECM) and amorphous masses of mesangial matrix. The local exaggeration of these deposits results in the formation in the typical diabetic nodule. To clarify the composition of the ECM of sclerotic lesions in DN, we investigated the distribution of type III and type IV collagens and their mRNAs by immunohistochemistry and in situ hybridization, respectively. In normal renal tissues, there was no intraglomerular immunostaining for type III collagen, while strongly positive staining was found in the extraglomerular interstitum. Positive immunostaining for type IV collagen was also present in the mesangium, glomerular basement membrane (GBM), Bowman's capsule, and the vascular pole of the normal glomerulus. In DN, the nodular lesions were negative for type III collagen and strongly positive for type IV collagen. On the other hand, in the late stage of global sclerosis, both type III and type IV collagens were diffusely present in the sclerotic matrix. To determine the origins of these type III and type IV collagens in the sclerotic matrix, in situ hybridization was performed, utilizing thymine-thymine (T-T) dimerized synthetic oligonucleotides complementary to either proα(III) chain or pro α1 (IV) chain mRNAs as probes. The signals were detected by enzyme immunohistochemistry using an anti-T-T antibody. Intraglomerular cells (glomerular epithelial and mesangil cells) containing type III collagen mRNA were found in DN with sclerotic lesions, but not in normal glomeruli. At this stage of sclerosis, intraglomerular cells (mainly glomerular epithelial cells and infrequently mesangial cells) were positive for type IV collagen mRNA, but there were few positive cells in globally sclerotic glomeruli. This study provides evidence that both type III and type IV collagens are synthesized by intraglomerular cells during sclerosis and become significant constituents of the sclerotic matrix in DN.     

Fibrinogen-like protein 2 expression correlates with microthrombosis in rats with type 2 diabetic nephropathy

Fibrinogen-like protein 2 (fgl2),a novel prothrombinase,is involved in microthrombosis.We examined fgl2 expression in the glomerular and tubulointerstitial capillaries and its correlation with microthromsis in rats with streptozocin-induced type 2 diabetic nephropathy.Our RT-PCR and immunoblotting analysis showed that fgl2 mRNA and protein levels were increased in microvascular endothelial cells of the glomeruli and renal interstitia at week 19 and became significantly elevated with the development of diabetic nephropathy (P < 0.01).Fgl2 was not or only weakly expressed in the renal tissues of normal rats.Furthermore,a direct significant correlation (r=0.543,P < 0.01) was found between fgl2 expression and microthrombotic capillaries in the renal tissues.Enzyme linked immunosorbent assays (ELISA) additionally showed that circulating TNF-α levels in rats with type 2 diabetes were significantly elevated and closely correlated with fgl2 expression (r=0.871,P < 0.01).Our results suggest that fgl2 may activate renal microthrombosis,thus contributing to glomerular hypertension and renal ischemia.     

厄洛替尼减轻STZ诱导的糖尿病肾病模型大鼠的肾损伤

目的:研究表皮生长因子受体(EGFR)抑制剂厄洛替尼(erlotinib)对糖尿病肾病大鼠肾脏的保护作用及机制。方法:采用大剂量(55 mg/kg)链脲佐菌素(STZ)腹腔注射诱导大鼠糖尿病肾病模型,以1周后血糖值16.7 mmol/L的大鼠为造模成功的标准。将糖尿病大鼠随机分为2组[STZ组和STZ+erlotinib(100 mg·kg~(-1)·d~(-1))组],并以正常大鼠为对照组(control组)。Erlotinib处理4周后,检测大鼠空腹血糖、血清肌酐和24 h尿蛋白含量的变化;HE染色和Masson染色观察肾脏组织病理学改变;Western blot检测各组肾脏组织中EGFR、p-EGFR、转化生长因子β1(TGFβ1)、Smad2/3、p-Smad2/3、Ⅳ型胶原蛋白(ColⅣ)和纤连蛋白(fibronectin)的蛋白水平;活性氧簇(ROS)和丙二醛(MDA)试剂盒分别检测各组肾脏组织中ROS和MDA水平。结果:与control组相比,STZ组血糖、24 h尿蛋白和血清肌酐水平均显著升高(P0.01),肾组织形态学出现异常变化;与STZ组相比,STZ+erlotinib组的血糖、24 h尿蛋白水平和血清肌酐水平显著降低(P0.05),肾小球结构恢复正常,肾小球系膜细胞增生程度明显减弱。厄洛替尼明显抑制了STZ大鼠肾组织中p-EGFR、TGFβ1、p-Smad2/3、ColⅣ和fibronectin蛋白水平,也明显抑制了STZ大鼠肾组织中ROS和MDA水平。结论:厄洛替尼可能通过抑制EGFR/TGFβ1-Smad2/3信号通路的激活来抑制糖尿病肾病肾组织的纤维化和氧化应激反应,从而减轻肾损伤。     

Renoprotection: focus on TRPV1, TRPV4, TRPC6 and TRPM2

Members of the transient receptor potential (TRP) cation channel receptor family have unique sites of regulatory function in the kidney which enables them to promote regional vasodilatation and controlled Ca²⁺ influx into podocytes and tubular cells. Activated TRP vanilloid 1 receptor channels (TRPV1) have been found to elicit renoprotection in rodent models of acute kidney injury following ischaemia/reperfusion. Transient receptor potential cation channel, subfamily C, member 6 (TRPC6) in podocytes is involved in chronic proteinuric kidney disease, particularly in focal segmental glomerulosclerosis (FSGS). TRP vanilloid 4 receptor channels (TRPV4) are highly expressed in the kidney, where they induce Ca²⁺ influx into endothelial and tubular cells. TRP melastatin (TRPM2) non‐selective cation channels are expressed in the cytoplasm and intracellular organelles, where their inhibition ameliorates ischaemic renal pathology. Although some of their basic properties have been recently identified, the renovascular role of TRPV1, TRPV4, TRPC6 and TRPM2 channels in disease states such as obesity, hypertension and diabetes is largely unknown. In this review, we discuss recent evidence for TRPV1, TRPV4, TRPC6 and TRPM2 serving as potential targets for acute and chronic renoprotection in chronic vascular and metabolic disease.     

血浆型凝溶胶蛋白和纤连蛋白在糖尿病肾病肾组织的表达及其临床病理资料分析

目的 对血浆型凝溶胶蛋白(pGSN)和纤连蛋白(FN)在糖尿病肾病(DN)患者肾组织的表达及其临床病理学资料进行分析.方法 根据临床诊断,将肾活检诊断为DN的67例患者分为4组:DN或糖尿病(DM)组、DM或DN合并高血压组、DM或DN合并其他肾脏病组和单纯诊断为其他肾脏疾病组,而后者又可分为血糖正常和血糖升高两种.利用免疫组织化学方法检测pGSN和FN在上述组织的石蜡切片的表达情况,与临床资料结合进行统计学分析.结果 pGSN和FN主要表达在肾小球和肾小管中,尤其在纤维化的肾小球和肾小管表达最为明显,两者均与肾小球的硬化率(硬化肾小球/总肾小球数)相关,而与其他的临床指标没有明显相关性.在四组临床诊断分组中,临床DM症状不典型伴有肾脏改变且血糖升高组,其肾小球滤过率(eGFR)明显降低(P＜0.05).而血糖正常组,其凝血酶原时间(PT)、血糖、丙氨酸转氨酶(ALT)、二氧化碳结合力及糖化血红蛋白相较于其他组有统计学意义(P＜0.05).而DM或DN伴有高血压组的患者其凝血项检查中的凝血酶原时间活动度(PT％)、凝血酶原时间国际标准化比值(PTINR)、凝血酶时间(TT)的差异有统计学意义(P＜0.05),ATL、天冬氨酸氨基转移酶(AST)明显降低(P＜0.05).结论 pGSN和FN反映肾小球的纤维化程度.临床上不能明确诊断为DN的病人,肾活检确诊时其肾功能的损害更严重.伴有高血压的DN患者,其凝血系统及肝功发生明显变化.     

2型糖尿病患者血IL-6与循环内皮细胞的关系及意义

目的探讨2型糖尿病(DM)患者血管内皮细胞损伤与血白介素6(IL-6)的关系及其意义。方法检测45例2型DM患者及20例正常人外周血IL-6和循环内皮细胞(CEC)水平并进行比较。结果①2型DM患者血IL-6和CEC水平高于正常人(P<0.05);②早期糖尿病肾病患者血CEC水平明显高于单纯糖尿病患者(P<0.01);③多元逐步回归分析显示CEC与血IL-6水平和尿蛋白排泄率(UAER)显著相关,标准偏回归系数β分别为0.264(P=0.033)和0.545(P=0.000)。结论2型糖尿病血管内皮细胞损伤与体内IL-6升高有密切关系,并在糖尿病肾病的发病过程中起一定作用。     

糖尿病肾病患者血清IL-1、IL-6含量测定

目的：检测糖尿病肾病患者血清白介素-1、白介素-6的水平,为进一步探讨与糖尿病肾病的关系提供资料。方法：2型糖尿病69例,尿白蛋白排泄率（UAE）在20-200 μg/min的21例患者为DN₁组,UAE>200 μg/min 22例患者为DN₂组,UAE<20 μg/min的26例为DM组,正常对照组20例,用免疫放射法测定血清IL-1、IL-6水平。结果：DN₂组血清IL-1含量为(31.23±6.69) ng/L,高于正常对照组[(21.37±8.24) ng/L]及DM组[(26.45±7.19) ng/L)], P<0.01, P<0.05。DM组血清IL-6含量为(1.50±0.79) μg/L,明显高于对照组（P<0.01）,DN₁组含量(1.02±0.77) μg/L,DN₂组为(0.97±0.67) μg/L,均低于DM组（两者P<0.05）。结论：糖尿病合并肾病患者血清IL-1水平高于正常对照及DM组,血清IL-6低于DM组提示其与糖尿病肾病的发病可能有一定的关系。     

Requirement for TLR2 in the development of albuminuria,inflammation and fibrosis in experimental diabetic nephropathy

Inflammation and fibrosis are essential elements of diabetic nephropathy (DN). We tested the hypothesis that these elements are dependent upon Toll-like receptor 2 (TLR2) signalling by examining WT and TLR2^-/- mice in an experimental model of DN. Diabetes was induced in WT and TLR2^-/- mice by i.p. injection of streptozotocin. Kidney injury was assessed at 6, 12 and 24 weeks after induction of diabetes. Gene expression of TLR2, its endogenous ligands and downstream cytokines, chemokines and fibrogenic molecules were upregulated in kidneys from WT mice with streptozotocin diabetes. TLR2^-/- mice were protected against the development of DN, exhibiting less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic WT controls. Marked reductions in interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) were also evident in TLR2 deficient mice. Consistent with our in vivo results, high glucose directly promoted TLR2 activation in podocytes and tubular epithelial cells (TECs) in vitro, resulting in NF-κB activation, inflammation and TGF-β production. We conclude that TLR2 was required for the full development of inflammation, kidney damage and fibrosis in this model of DN. As TLR2 is known to be expressed by intrinsic kidney cells and as high concentration glucose stimulated podocytes and TECs in vitro to express TLR2 and TLR2 ligands, pro-inflammatory and pro-fibrotic cytokines in a TLR2 dependent manner in the present study, it appears likely that TLR2 signalling in intrinsic kidney cells contributes to the pathogenesis of diabetic nephropathy.