调节内质网应激对糖尿病小鼠肾脏SET7/9表达的影响

目的 探讨调节内质网应激对小鼠肾组织组蛋白甲基转移酶(HMT) SET7/9表达的影响及意义.方法 db/db小鼠按随机数字表法分为糖尿病肾病(DN)组和甜菜碱治疗(DN+B)组;db/m小鼠作为正常对照(NC)组,每组各18只.实验第4、8、12周末分别采用实时定量PCR和(或)Western印迹法测定小鼠肾组织SET7/9、葡萄糖调节蛋白(GRP)78、H3K4me2和单核细胞趋化蛋白1(MCP-1)表达水平;ELISA法测定24 h尿蛋白排泄率(UPER)和尿MCP-1浓度;全自动生化分析仪检测血糖(BG)、血肌酐、血尿素氮的动态改变;PAS染色观察肾脏病理改变.结果 与NC组比较,DN组BG、BUN、UPER、MCP-1均显著升高(均P＜0.05),且呈时间依赖性.DN组小鼠第4周末开始出现肾小球基底膜增厚、系膜细胞增生改变,第12周末出现明显系膜基质积聚.与NC组比较,DN组肾组织GRP78、SET7/9的mRNA和蛋白质表达水平均显著升高,H3K4me2蛋白水平也显著升高,且呈时间依赖效应.与DN组比较,甜菜碱治疗组小鼠肾小球病变明显减轻,GRP78、SET7/9的mRNA及蛋白表达水平显著降低,BG、BUN、UPER、MCP-1、H3K4me2水平显著降低(均P＜0.05).结论 内质网应激可能是介导糖尿病小鼠肾脏SET7/9表达的上游机制.     

自发性2型糖尿病KKAy小鼠肾小球microRNA表达谱和氯沙坦治疗效应

目的 分析糖尿病肾病(DN)发病过程中肾小球miRNA表达谱的变化,观察血管紧张素受体拮抗剂(ARB)氯沙坦对DN肾小球miRNA表达谱的影响,确认在DN发病过程中发挥关键作用的miRNA.方法 8周龄KKAy小鼠随机分为氧沙坦治疗组(10 mg·kg-1·d-1)和非治疗组,C57BL/6小鼠作为正常对照组.于20周龄检测体质量、随机血糖、尿微量白蛋白、尿肌酐,观察肾脏形态改变.应用磁珠灌注法分离肾小球,提取总RNA,应用Affymetrix GeneChip miRNA芯片,分析KKAv小鼠肾小球microRNA表达谱的变化,以及氯沙坦对microRNA表达谱的影响.结果 KKAy小鼠的体质量和血糖较正常对照C57BL/6组小鼠显著升高(均P＜ 0.05),氯沙坦治疗显著改善2型糖尿病KKAy小鼠的尿白蛋白/肌酐比值[( 539.71±100.23) mg/g比(728.00±177.19) mg/g,P＜0.05]和肾脏病理损害,而对血糖无影响.miRNA芯片分析结果发现,与正常对照C57BL/6小鼠相比,20周龄KKAy小鼠肾小球内10个miRNA的表达上调;12个miRNA的表达下调.与KKAy非治疗组小鼠相比,20周龄氯沙坦治疗组KKAy小鼠肾小球内共有4个miRNA表达下调,其中miR-503和miR-181d在KKAy非治疗组小鼠肾小球内的表达显著上调,氯沙坦治疗可抑制其过表达.结论 miR-503和miR-181d在糖尿病KKAy小鼠肾小球内的表达显著上调,氯沙坦治疗可抑制其在糖尿病状态下的异常表达,可能为糖尿病肾病新的治疗靶点.     

甘草次酸对糖尿病肾病动物模型的疗效观察

目的探讨甘草次酸(GA)对于糖尿病肾病(DN)的治疗效果。方法利用CS7 BL/6背景的小鼠和db/db小鼠分别构建1型和2型DN的动物模型。将C57 BL/6小鼠分别分为正常对照组(Con组)、甘草次酸处理的对照组(Con+GA组)、DN组和甘草次酸处理的DN组(DN+GA组),db/db小鼠分为对照组、DN组和治疗组。然后分别观察GA处理对于小鼠血糖、体质量、尿肌酐、尿蛋白/肌酐的比值、细胞外基质的沉积以及细胞凋亡的影响。结果和Con组的血糖相比,DN组血糖水平和尿蛋白/肌酐的比值显著升高,差异有统计学意义(P0.01);细胞外基质的沉积和细胞凋亡增加;尿肌酐的排泄显著减少,差异有统计学意义(P0.01)。和DN组相比,DN+GA组小鼠尿肌酐的水平以及尿蛋白/肌酐的比值有显著性差异(P0.01);细胞外基质的沉积明显缓解;细胞凋亡水平下降。1型DN动物模型中,Con组和DN组小鼠体质量差异有显著性(P0.05),同时DN组和DN+GA组小鼠体质量差异有显著性(P0.01)。结论 GA对于1型以及2型DN动物模型中肾脏的病变均能够发挥保护作用,从而延缓肾脏疾病的进展。     

高迁移率族蛋白1对狼疮肾炎小鼠肾小球细胞增殖的影响

目的 通过敲低肾组织高迁移率族蛋白1(HMGB1)表达,探讨其对改善狼疮肾炎小鼠肾功能,降低肾小球细胞增殖水平的影响.方法 MRL/Faslpr鼠(n=24)被随机分为模型组、shHMGB1组和空质粒组;选取周龄、体质量相匹配的MRL/MpJ鼠为健康对照组.shHMGB1组和空质粒组采用电穿孔转染技术分别转染shHMGB1质粒和空质粒,模型组和健康对照组仅转染生理盐水.用全自动生化分析仪检测小鼠血清尿素氮和肌酐水平,测定尿蛋白浓度并计算24 h尿蛋白量(UP).HE染色观察肾组织的形态学表现;免疫荧光和Western印迹法检测小鼠肾小球中HMGB1和增殖细胞核抗原(PCNA)的表达;实时定量PCR法检测小鼠肾小球HMGB1和PCNA mRNA表达变化.结果 (1)与健康对照组相比,模型组小鼠肾小球HMGB1 mRNA和蛋白的表达升高(均P＜0.05);与模型组相比,shHMGB1组小鼠肾小球中HMGB1 mRNA和蛋白的表达降低(均P＜0.05).(2)与模型组相比,shHMGB1组小鼠尿蛋白减少(P＜0.05).(3)免疫荧光和Western印迹结果显示,与健康对照组相比,模型组小鼠肾小球中PCNA mRNA和蛋白表达升高(P＜0.05).与模型组相比,shHMGB1组肾小球中PCNA表达降低(P＜0.05).结论 敲低肾组织HMGB1表达可改善狼疮肾炎小鼠的肾功能,降低肾小球细胞的增殖水平.     

下调miR-153促进高糖诱导的肾小管上皮细胞-间充质转化

目的：探讨miR-153调控高糖诱导肾小管上皮细胞（HK2）-间充质转化（EMT）的机制,为研究糖尿病肾病（DN）间质纤维化机制提供新思路。方法：采用生物信息学方法预测调控Snail的候选miRNAs;在db/db小鼠肾组织中验证所有候选miRNAs的表达并分别与Snail做pearson相关性分析,筛选出与Snail负相关最显著的miRNA;以高糖诱导的HK2细胞为载体,转染该miRNA mimics 72 h后,采用real-time PCR、western blot方法,观察该miRNAs、Snail以及E-cadherin的表达改变。结果：调控Snail的候选miRNAs共7个,分别是：miR-153、miR-30e、miR-30d、miR-30b、miR-384-5p、miR-30a、miR-30c;与db/m相比,miR-153、miR-30d及miR-30e在db/db小鼠肾皮质中表达显著下调（P〈0.05）,其中miR-153与Snail表达水平负相关最显著（r=-0.501 56）;高糖诱导HK2细胞下调miR-153,肾小管上皮细胞标志蛋白E-cadherin表达减少;过表达miR-153抑制HK2细胞表达Snail,而E-cadherin水平增加。结论：MiR-153可能通过靶基因Snail参与调控高糖诱导的HK2细胞EMT。     

脂联素基因重组慢病毒对早期糖尿病肾病小鼠肾脏的保护作用

目的 探讨静脉注射小鼠脂联素基因重组慢病毒对糖尿病肾病(DN)模型小鼠的肾脏保护作用及其机制.方法 40只C57BL/6小鼠按数字随机法分为正常对照组(NC组)、糖尿病肾病组(DN组)、阴性对照病毒(Lenti-IRES-EGFP)治疗组(DL组)、脂联素基因重组慢病毒( Lenti-Acdc-IRES-EGFP)治疗组(DA组),每组10只.应用链脲菌素(SFZ)结合高脂饮食诱导建立DN模型.重组慢病毒注射8周后,检测各组小鼠血浆脂联素水平、肾功能、尿白蛋白排泄率、肾组织病理改变.用免疫组织化学法原位检测肾组织增殖细胞核抗原( PCNA)表达.用Western印迹检测肾组织腺苷酸活化蛋白激酶α(AMPKα)、哺乳动物雷帕霉素靶蛋白( mTOR)水平.结果 成功构建脂联素超表达DN动物模型.与NC组比较,第12周末DA组小鼠肾质量指数(肾质量/体质量,KWI)、平均肾小球体积(MGV)、系膜面积比(FMA)、24h尿蛋白(UTP)均明显升高(P＜0.05),但低于DN组、DL组(P＜0.05).DA组肾组织PCNA阳性细胞数显著低于DN组、DL组(P＜0.01).与DN组、DL组相比,DA组小鼠肾组织AMPKα磷酸化水平显著升高(P＜0.01),mTOR磷酸化水平降低(P＜0.01).结论 脂联素通过激活AMPK信号通路,抑制mTOR信号活化,进而抑制早期DN时肾组织异常增殖.     

IGF-1R抑制剂对糖尿病肾病小鼠肾间质巨噬细胞浸润、lncRNA-Gm4419表达的影响

目的:探究胰岛素样生长因子1(IGF-1R)受体抑制剂对糖尿病肾病小鼠肾间质巨噬细胞浸润、lncRNA-Gm4419表达的影响。方法:选取60只雄性C57BL/6小鼠,采用随机数字表法分为:空白对照组、模型组和抑制剂组,每组20只。采用腹腔注射链脲佐菌素制成糖尿病肾病(DN)小鼠模型;采用免疫组化和流式细胞仪检测各组小鼠肾间质中CD~+_(68)细胞、F4/80~+细胞数目;采用蛋白质印迹检测各组小鼠肾脏组织中细胞因子信号转导抑制因子2(SOCS2)、Toll样受体4(TLR4)和IGF-1R的表达;采用RT-PCR检测各组小鼠肾组织中lncRNA-Gm4419及IGF-1R mRNA表达情况。结果:相比空白对照组,模型组小鼠CD~+_(68)细胞数目、F4/80~+细胞数目、TLR4蛋白相对表达、IGF-1R蛋白相对表达、Gm4419基因相对表达和IGF-1R9基因相对表达均显著升高,SOCS2蛋白相对表达水平显著降低(P0.05);相比模型组,抑制剂组小鼠CD~+_(68)细胞数目、F4/80~+细胞数目、TLR4蛋白相对表达、IGF-1R蛋白相对表达、Gm4419基因相对表达和IGF-1R9基因相对表达均显降低高,SOCS2蛋白相对表达水平显著升高(P0.05)。结论:抑制IGF-1R的表达可以有效抑制糖尿病肾病小鼠肾间质巨噬细胞浸润和lncRNA-Gm4419表达,其作用机制可能与促进SOCS2表达调控相关信号通路相关。     

阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用

目的 观察肾素抑制剂阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用。 方法 8周龄db/db和db/m小鼠行单侧肾切除,16周进入实验,分为4组：db/m小鼠对照组（db/m组）、db/db糖尿病小鼠对照组（db/db组）、db/db糖尿病小鼠阿利吉仑3 mg&#8226;kg-1&#8226;d-1治疗组（db/db+A3组）和db/db糖尿病小鼠阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗组(db/db+A25组)。阿利吉仑溶于磷酸盐缓冲液（PBS,350 mg/L）皮下泵入（0.25 μl/h）给药,疗程４周。治疗前后检测体质量、血糖、糖化血红蛋白、尿蛋白量、血压水平;PAS染色观察肾脏组织学变化;ELISA法检测肾皮质转化生长因子β1（TGF-β1）和纤溶酶原激活抑制因子1（PAI-1）含量;间接免疫荧光检测肾小球Ⅳ型胶原（ColⅣ）和纤连蛋白（FN）表达;实时定量PCR检测TGF-β1、PAI-1、ColⅣ、FN和肾素mRNA表达;放射免疫法检测肾皮质肾素活性和血管紧张素Ⅱ（AngⅡ）水平。 结果 与db/m组小鼠比较,db/db组小鼠有大量蛋白尿,肾小球细胞外基质沉积增加,TGF-β1、PAI-1、ColⅣ和FN蛋白及mRNA表达增加,同时肾皮质肾素mRNA、肾素活性和AngⅡ水平增高（均P < 0.05）。阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗在没有影响血压情况下,显著减少db/db小鼠24 h尿蛋白量,减少肾小球细胞外基质沉积,减少TGF-β1、PAI-1、ColⅣ、FN蛋白和mRNA表达,同时降低肾皮质肾素活性和AngⅡ水平（均P < 0.05）。 结论 阿利吉仑对2型糖尿病db/db小鼠肾脏损伤有保护作用。     

胃癌组织/细胞中miR-429和ZEB1/2表达的相关性及临床意义

目的:探讨胃癌组织及胃癌细胞中miR-429和E盒结合锌指蛋白(ZEB)1/2表达的相关性,及其与临床病理参数的关系。方法:收集胃癌手术标本,分别采用Western blot法和定量RT-PCR法检测胃癌组织及胃癌细胞中ZEB1/2及miR-429的表达,分析两者表达相关性及其与临床病理参数的关系。将miR-429表达及as-miR-429质粒转染胃癌细胞,再将ZEB1/2-3'UTR荧光素酶报告质粒转染细胞,测定荧光素酶在细胞中的表达。结果:胃癌组织中ZEB1和ZEB2表达明显高于癌旁组织,而miR-429表达低于癌旁组织(P0.05)。miR-429与ZEB1/2表达均呈显著负相关(r=-0.65、-0.58,P0.0001)。miR-429与ZEB1/2 mRNA的3'-UTR结合,抑制ZEB蛋白翻译表达。胃癌组织中miR-429和ZEB1/2的表达水平均与脉管瘤栓相关(P0.05),而与患者的性别、年龄、淋巴结转移、TNM分期无关(P0.05)。结论:miR-429在胃癌发生、进展过程中发挥重要作用,可通过其与下游关键调节因子,如ZEB1/2的协同作用而实现。     

MiR-26b通过靶基因GSK3β调控高糖诱导的系膜细胞肥大

目的:研究miR-26b调控高糖诱导系膜细胞(MCs)肥大的机制,为其在糖尿病肾病(DN)早期发病机制研究提供新线索。方法:采用生物信息学方法预测调控GSK3β的候选miRNAs;在db/db小鼠肾皮质中验证所有候选miRNAs的表达并分别与GSK3β做pearson相关性分析;以高糖诱导的MCs为载体,采用real-time PCR,观察与GSK3β显著负相关的miRNAs和GSK3β表达水平;转染miR-26b抑制子后,检测高糖诱导的GSK3β以及α-SMA的表达改变。结果:预测到调控GSK3β的候选miRNAs共9个:分别为miR-199a-5p,miR-128,miR-23b,miR-23a,miR-29a,miR-29c,29b,miR-26b,miR-26a;与对照组相比,miR-128,miR-23b,miR-26b,miR-29a,26b在db/db小鼠肾皮质中表达显著上调;其中miR-26b与GSK3β表达水平负相关最显著(r=-0.470 29);高糖诱导的MCs中miR-26b与GSK3β亦呈现显著负相关,miR-26b抑制子部分逆转GSK3β表达水平,同时α-SMA、Ⅳ型胶原表达水平上调。结论:miR-26b可能通过其靶基因GSK3β参与高糖诱导的MCs肥大机制的调控,为DN早期发病机制研究提供了新的思路。     

Expression and role of microRNA-155 in db/db mice

Objective To investigate the expression of microRNA-155 (miR-155) in serum and kidney of C57BLKS/db (db/db) mice and its role in the pathogenesis of diabetic kidney disease (DKD). Methods The db/db mice (n=24) were divided into 6, 8, and 10 weeks old groups (n=8) with age increasing according to the random number table, and C57BL/6 mice of the same age were used as control group. The expression of miR-155 in mouse serum and kidney tissue was determined using real-time quantitative PCR. The mRNA and protein expression of Ets-1, eNOS, AGTR1 in renal tissues was verified by real-time quantitative PCR, Western blotting and immunohistochemistry. Results Compared with the control group, the expression of miR-155 in serum of db/db mice at 6, 8 and 10 weeks of age were significantly increased (all P＜0.01), and the increase of miR-155 was most obvious at 10 weeks of age (P＜0.01). Meanwhile the expression of miR-155 in kidney tissues of 6, 8 and 10 weeks old db/db mice was significantly up-regulated (all P＜0.01), and the highest expression of miR-155 was at 10 weeks of age (P＜0.01). Immunohistochemistry showed that Ets-1, eNOS and AGTR1 were localized in glomerular endothelial cells. The results of real-time quantitative PCR showed that the mRNA expression of Ets-1, eNOS and AGTR1 were down-regulated in the kidney tissues of db/db mice at 6, 8 and 10 weeks of age compared to the control(all P＜0.05), and the level of down-regulation was the most obvious at 10 week. Western blotting results showed that there was no significant change in Ets-1, eNOS and AGTR1 in 6-week-old db/db mice compared to the control group; the eNOS protein expression was down-regulated at 8 weeks of age (P＜0.05); the expression of AGTR1 protein was down-regulated (P＜0.05), and the protein expression of Ets-1 and eNOS were significantly down-regulated at 10-week age (both P＜0.01). Conclusions The expression of miR-155 in serum and kidney tissues of db/db mice increases during the progression of DKD, while the expression of miR-155 target genes Ets-1, eNOS and AGTR1 decreases with the progression of DKD. MiR-155 may participate in the development and progression of DKD by inhibiting its target genes Ets-1, eNOS and AGTR1, affecting endothelial cell function.     

PPARalpha agonist fenofibrate improves diabetic nephropathy in db/db mice

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the ligand-activated nuclear receptor superfamily, and plays an important role in lipid metabolism and glucose homeostasis. The purpose of this study is to determine whether the activation of PPARalpha by fenofbrate would improve diabetes and its renal complications in type II diabetes mellitus. Male C57 BLKS db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet chow (db/db, n=8; db/m, n=6) or a diet containing fenofibrate (db/db, n=8; db/m, n=7). Mice were followed for 8 weeks. Fenofibrate treatment dramatically reduced fasting blood glucose (P<0.001) and HbA1c levels (P<0.001), and was associated with decreased food intake (P<0.01) and slightly reduced body weight. Fenofibrate also ameliorated insulin resistance (P<0.001) and reduced plasma insulin levels (P<0.05) in db/db mice. Hypertrophy of pancreatic islets was decreased and insulin content markedly increased (P<0.05) in fenofibrate-treated diabetic animals. In addition, fenofibrate treatment significantly reduced urinary albumin excretion (P<0.001). This was accompanied by dramatically reduced glomerular hypertrophy and mesangial matrix expansion. Furthermore, the addition of fenofibrate to cultured mesangial cells, which possess functional active PPARalpha, decreased type I collagen production. Taken together, the PPARalpha agonist fenofibrate dramatically improves hyperglycemia, insulin resistance, albuminuria, and glomerular lesions in db/db mice. The activation of PPARalpha by fenofibrate in mesangial cells may partially contribute to its renal protection. Thus, fenofibrate may serve as a therapeutic agent for type II diabetes and diabetic nephropathy.     

Regulation of melatonin on Toll-like receptor 4 signaling in diabetic db/db mice kidneys

Objective To investigate the regulation of melatonin (MT) on Toll-like receptor 4 (TLR4) signaling in diabetic db/db mice kidneys. Methods The 48 10-week-old male db/db mice were randomly divided into db/db group, db/db+MT 50 μg/kg group, db/db+MT 100 μg/kg group and db/db+MT 200 μg/kg group, each consisting of 12 mice. These mice received i.p. injections of MT These mice received i.p. injections of MT [dissoved in phosphate buffer solution (PBS)/ dimethylsulfoxide (DMSO) solution, given every day]. Alternatively, 12 db/m mice served as the control group. db/m and db/db group were injected i.p. with the same volume of PBS/DMSO solution. The animals were sacrificed after 12 weeks of dosage administration. Blood glucose (BG), body weight (BW), kidney weight (KW) and 24 h urinary albumin excretion rate (UAER) were determined; Kidney pathological lesions were evaluated by renal pathological staining. Immunohistochemistry of renal TLR4, NF-κB p65, and ED-1 was performed to determine the immunoreactivity. Western blotting was used to detect the expression of renal TLR4, myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF-3) and NF-κB p65, while the mRNA expressions of renal tumor necrosis factor -α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were evaluated by real-time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were much higher in db/db mice group (P＜0.01), while KW in db/db+MT (100, 200 μg/kg) groups and UAER level in db/db+MT (50, 100, 200 μg/kg) groups were distinctly decreased compared with those in db/db group (P＜0.01). In week 12 db/db mice, the glomerular mesangial expansion index and tubulointerstitial injury index were increased compared with those in db/m mice (P＜0.01). The above kidney histopathologic lesions were distinctly ameliorated by 50, 100, 200 μg/kg MT (P＜0.05). Immunohistochemistry intensity of renal TLR4, NF-κB p65 and ED-1 displayed obvious differences between db/m mice and db/db mice (P＜0.01), and that were remarkably decreased in db/db+MT (50, 100, 200 μg/kg) mice compared with db/db mice (P＜0.05). Western blotting showed that the protein expression of renal TLR4, MyD88, TRIF, IRF-3 and NF-κB p65 were stronger in db/db group compared with those in db/m group (P＜0.05) and weaker in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.05). Futhermore, the mRNA expressions of renal MCP-1 and TNF-α were higher in db/db group compared with those in db/m group (P＜0.01) and lower in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.01). Conclusion Melatonin may partly down-regulate TLR4 signaling pathway to inhibit Inflammatory reaction and alleviate kidney injury in diabetic db/db mice.     

Prevention and reversal of diabetic nephropathy in db/db mice treated with alagebrium (ALT-711)

BACKGROUND: Alagebrium (ALT-711) has been shown to improve renal dysfunction in animal models of diabetes. METHODS: To test its effects in diabetic nephropathy (DN), ALT-711 was administered (1 mg/kg daily i.p.) to 9-week-old female db/db mice (n = 15, group A1) for 3 weeks and to 3-month-old (n = 15, group A2), 7-month-old (n = 7, group A3), and 12-month-old (n = 5, group A4) female db/db mice for 12 weeks, while a similar number of diabetic and nondiabetic mice were used as controls. The epsilonN-carboxymethyllysine (CML) levels in serum, urine, skin, and kidney tissue were measured by enzyme-linked immunosorbent assay. The renal morphometric parameters were assessed by electron and light microscopy. RESULTS: By the 3rd week of treatment, the serum CML level decreased by 41%, and the urinary CML concentration increased by 138% from baseline, while the urinary albumin/creatinine ratio was lower (p < 0.05) in diabetic and nondiabetic group A1 mice. After 3 months of treatment, serum, skin, and kidney CML levels and urinary albumin/creatinine ratio were lower (p < 0.05) and the urinary CML levels higher (p < 0.05) in treated group A2, A3, and A4 animals compared with groups which received phosphate-buffered saline, with a similar pattern observed in nondiabetic mice. The renal morphological parameters characteristic of DN decreased in treated compared with untreated mice. CONCLUSION: Alagebrium may prevent, delay, and/or reverse established DN in db/db mice by reducing the systemic advanced glycation end product pools and facilitating the urinary excretion of advanced glycation end products.     

Inhibiting albumin glycation ameliorates diabetic nephropathy in the db/db mouse

Albumin modified by Amadori glucose adducts stimulates the expression of extracellular matrix proteins by glomerular mesangial and endothelial cells, and has been mechanistically linked to the pathogenesis of diabetic nephropathy. To test the hypothesis that inhibiting the formation of glycated albumin might beneficially influence the development of kidney disease in diabetes, we treated diabetic db/db mice for 12 weeks with a low-molecular-weight compound (EXO-226) that impedes the condensation of free glucose with lysine epsilon-amino groups in albumin. Administration of EXO-226 (3 mg/kg) twice daily by gavage normalized the plasma concentration of glycated albumin within days after initiation of treatment and maintained glycated albumin within the normal range throughout the study, despite persistent and severe hyperglycemia. Urine albumin excretion, which was markedly increased at the start of the study (age 12 weeks), was significantly reduced in treated diabetic animals compared with their untreated diabetic littermates. The fall in creatinine clearance that was observed in untreated diabetic animals was prevented in diabetic littermates that received treatment. Compared with the nondiabetic controls, the amount of glomerular mesangial matrix was threefold greater in untreated diabetic mice; in contrast, the mesangial matrix fraction was only 1. 5 times that of nondiabetic controls in the treated diabetic animals, representing a reduction in mesangial matrix accumulation of more than 50%. EXO-226 also reduced the overexpression of mRNA encoding for alpha1 (IV) collagen in renal cortex of db/db mice. We conclude that normalization of plasma glycated albumin concentrations with the glycation inhibitor EXO-226 ameliorates the glomerular structural and functional abnormalities associated with diabetic nephropathy in the db/db mouse.     

Disparate phospho-Smad2 levels in advanced type 2 diabetes patients with diabetic nephropathy and early experimental db/db mouse model

Uncontrolled activation of transforming growth factor beta (TGF-β) family members is hypothesized to participate in type 2 diabetes (T2D) dependent diabetic nephropathy (DN). We evaluated and compared downstream activation of the Smad2-signaling pathway in kidney samples from T2D patients to kidneys from the T2D model of leptin receptor deficient db/db mouse. Furthermore, expression of TGF-β family members was evaluated to elucidate molecular mechanisms in the mouse model. Kidney samples from patients with advanced stages of DN showed elevated pSmad2 staining whereas db/db mouse kidneys surprisingly showed a decrease in pSmad2 in the tubular compartment. Structurally, kidney tissue showed dilated tubules and expanded glomeruli, but no clear fibrotic pattern was found in the diabetic mice. Selective TGF-β family members were up-regulated at the mRNA level. Antagonists of bone morphogenetic protein (BMP) ligands, such as Gremlin1, USAG1 and Sclerostin, were strongly up-regulated suggesting a dampening effect on BMP pathways. Together, these results indicate a lack of translation from T2D patient kidneys to the db/db model with regards to Smad signaling pathway. It is plausible that a strong up-regulation of BMP antagonizing factors account for the lack of Smad1/5/8 activation, in spite of increased expression of several BMP members.