二十碳五烯酸对早期糖尿病肾病小鼠炎症和氧化应激状态的作用

目的：观察二十碳五烯酸（EPA）对早期糖尿病肾病动物模型KKAy/Ta小鼠炎症和氧化应激状态的作用。方法：KKAy/Ta小鼠随机分为两组：治疗组予以EPA1g·kg-1·d-1腹腔内注射共8周,对照组予以生理盐水注射。20周龄时检测表型变化和血清丙二醛（MDA）水平,及肾脏中巨噬细胞浸润、单核细胞趋化蛋白-1（MCP-1）、MDA和硝化酪氨酸（nitrotyrosine）表达的变化。结果：EPA显著降低了KKAy/Ta小鼠的血清三酰甘油和MDA水平,并减少尿白蛋白的排泄,改善了葡萄糖不耐受现象。EPA治疗后KKAy/Ta小鼠肾脏中MCP-1、巨噬细胞的浸润及MDA和nitrotyrosine表达明显减弱。结论：EPA改善了早期糖尿病肾病KKAy/Ta小鼠肾脏的炎症和氧化应激状态,其机制可能与EPA对代谢异常的纠正有关。     

早期应用低蛋白加α酮酸饮食对延缓KK—Ay小鼠糖尿病肾病进展的作用

目的：观察早期应用低蛋白加α酮酸饮食对2型糖尿病肾病动物模型KK—Ay小鼠肾脏病变的影响,并探讨其作用机制。方法：雄性KK—Ay小鼠30只,12周龄开始给予不同饮食干预,分为正常蛋白组（22％酪蛋白,NPD组）15只和低蛋白加α酮酸组（5％酪蛋白＋1％a酮酸,Keto组）15只;另设性别周龄相同的C57／BL6J小鼠15只为对照（CON组）,始终给予正常蛋白饮食。检测第8、14、20周龄各组小鼠尿白蛋白,血清中葡萄糖、胰岛素、糖化血红蛋白、胆固醇、三酰甘油、尿素氮、肌酐、白蛋白等生化变化。对20周龄小鼠肾组织病理进行图文分析和半定量检测,免疫组化方法测定肾组织中TGF—β．FN表达量。结果：Keto组小鼠14周龄时血糖、ACR（白蛋白／肌酐）、尿白蛋白排泄率、三酰甘油低于NPD组（P〈0．05）,20周龄时糖化血红蛋白、血胰岛素、肾小球面积及系膜区面积显著降低（P〈0．05）。免疫组化结果显示NPD组小鼠肾内TGF—β蛋白表达及肾小球FN沉积明显增多（P〈0．05）。结论：早期应用低蛋白加α酮酸饮食能够显著降低KK—Ay小鼠尿白蛋白,减轻系膜基质增生和结节性硬化,而不引起营养不良,并可能通过减少TGF-β表达抑制FN等系膜基质增加而延缓糖尿病肾病的进展。     

多配体蛋白聚糖4是糖尿病肾病的候选基因

目的 在2型糖尿病肾病(DN)白蛋白尿易感基因定位的基础上,进一步筛选白蛋白尿易感基因位点(UA-1)区域附近的候选基因.方法 提取20周龄雄性KK/Ta(n=3)和BALB/c (n=2)小鼠肾脏总RNA,应用Affymetrix Murine Genome U74Av2基因芯片检测肾脏基因表达谱.选择UA-1区域的差异表达基因多配体蛋白聚糖4(syndecan-4),竞争性RT-PCR验证基因芯片的结果.提取KK/Ta、BALB/c小鼠的基因组DNA,进行syndecan-4基因编码区和启动子区域的序列分析.结果 在2型糖尿病KK/Ta小鼠UA-1区域附近存在着约10个差异表达基因.其中syndecan-4在20周龄KK/Ta小鼠肾脏中的表达上调,为BALB/c小鼠的26.1倍.在syndecan-4基因编码区存在2个基因多态性,分别为A93C和T216C多态性,2者均为同义突变.在syndecan-4基因启动子区域存在3个基因多态性,分别为-T263C、-T396C 与-G669A多态性.TATA框位于转录起始位点上游321 bp处,-T263C处恰好为转录因子Clox 的结合位点.结论 syndecan-4基因位于2型糖尿病UA-1附近区域,在20周龄KK/Ta小鼠肾脏中的表达明显上调,是DN的候选基因.syndecan-4启动子处的基因多态性可能为其差异表达的原因.     

自发性2型糖尿病KK/Ta小鼠肾脏基因表达谱的研究

目的:应用基因芯片技术研究自发性2型糖尿病KK/Ta小鼠肾脏基因表达谱,旨在寻找糖尿病肾病的易感基因。方法:提取20周龄雄性KK/Ta(n=3)和BALB/c(n=2)小鼠肾脏总RNA,应用Affymetrix公司生产的Affymetrix Murine Genome U74Av2基因芯片检测肾脏基因表达谱。选择差异表达基因,通过竞争性RT-PCR反应验证基因芯片的结果。结果:98个已知基因和31个表达序列标签(ESTs)在20周龄KK/Ta与BALB/c小鼠的肾脏中存在着差异表达。与BALB/c小鼠相比,KK/Ta小鼠肾脏中21个已知基因和7个EST表达上调,77个已知基因和24个EST表达下调。竞争性RT-PCR反应确认了基因芯片研究的结果。结论:KK/Ta小鼠肾脏中的差异表达基因广泛参与细胞外基质的合成与降解、信号传导、转录调节与蛋白质合成、离子转运、葡萄糖与脂类代谢等。糖尿病肾病易感基因位点UA-1区域的差异表达基因S-腺苷高同型半胱氨酸水解酶基因Ahcy为糖尿病肾病的可能易感基因。     

清肝益肾祛风复方抗高血压肾损伤作用及机制研究

目的:探究清肝益肾祛风方对自发性高血压大鼠的降压、抗高血压肾损伤作用及其可能的作用机制,以期为临床治疗高血压肾损伤提供科学依据。方法:采用4周龄雄性SHR大鼠作为高血压模式动物,适应性饲养一周后随机分为SHR模型组、坎地沙坦治疗组,清肝益肾祛风方(QYQ)治疗组。正常对照组选用同周龄雄性WKY大鼠。从6周龄开始,QYQ治疗组和坎地沙坦治疗组每天分别给予2 ml的清肝益肾祛风方水煎液和坎地沙坦悬液灌胃。SHR组和WKY组采用同等体积的纯水做为对照,连续灌胃至20周龄。采用小动物无创血压计每2周测量一次血压;连续给药至20周龄后,10%水合氯醛腹1腔注射麻醉、腹主动脉取血后处死动物。留取双侧肾脏、采用离心机分离血清。左肾脱水固定包埋后做石蜡切片,Masson’s trichrome染色观察左侧肾脏组织病理变化;采用全自动生化检测仪检测大鼠血清中尿素、肌酐、尿酸水平; Realtime PCR方法检测右侧肾脏组织氧化应激标志物相关基因的mRNA表达水平、炎症因子基因的mRNA表达水平及胶原相关基因的mRNA表达水平。结果:与WKY组相比,SHR组大鼠血压从6周龄开始显著升高(P0.05),从12周龄开始达到稳定;与SHR组相比较,清肝益肾祛风方与坎地沙坦均能显著降低SHR大鼠血压(P0.05); Masson’s trichrome染色结果:WKY组肾脏组织未见病理损伤,SHR组大鼠肾脏组织出现较大面积纤维化。与SHR组相比,QYQ治疗组与坎地沙坦治疗组肾脏组织纤维化面积均显著降低(P0.05);生化检测结果提示:与WKY组相比,SHR组血清中尿酸、尿素含量显著升高(P0.05)。与SHR相比较,QYQ治疗组血清尿酸含量显著降低(P0.05)。Real-time PCR结果:与WKY组相比,SHR组肾脏组织氧化应激相关基因mRNA表达水平显著升高(P0.05),IL-23,TNF-α及TGF-β1等炎症因子相关基因的mRNA表达水平显著升高(P0.05),IL-10基因的mRNA表达水平显著降低(P0.05),胶原相关基因的mRNA表达水平显著上升(P0.05)。与SHR组相比,QYQ组肾脏组织氧化应激相关基因mRNA表达水平显著降低(P0.05),IL-23,TNF-α等炎症因子相关基因的mRNA表达水平显著降低(P0.05),IL-10的mRNA表达水平显著升高(P0.05),Col5a2及α-SMA基因的mRNA表达水平显著降低(P0.05)。结论:清肝益肾祛风方能显著降低SHR大鼠血压、显著减轻SHR大鼠肾脏组织纤维化,具有降压、抗高血压肾损伤的作用,其作用机制可能与减轻肾脏组织氧化应激反应、炎症反应及减少肾组织胶原纤维合成相关。     

渴络欣胶囊对早期糖尿病肾脏疾病肾小管的保护作用

目的 探究渴络欣胶囊对早期糖尿病肾脏疾病（diabetic kidney disease,DKD）肾小管的保护作用.方法 将符合纳入标准的45例早期DKD患者随机分为2组,渴络欣胶囊联合坎地沙坦酯为治疗组22例和坎地沙坦酯为对照组23例,疗程为2个月.比较2组治疗前、后尿中性粒细胞明胶酶相关脂质运载蛋白（NGAL）、尿β2微球蛋白（β2-MG）、尿白蛋白排泄率（UAER）、血肌酐（SCr）、空腹血糖（FBG）等指标变化.结果 治疗后2组尿NGAL、尿β2-MG、UAER较治疗前均有明显减少,差异有统计学意义（P＜0.05,P＜0.01）;治疗后尿NGAL、尿β2-MG减少,治疗组优于对照组,差异有统计学意义（P＜0.05）,但UAER减少,2组比较差异无统计学意义（P＞0.05）;2组FBG、SCr治疗前、后比较差异均无统计学意义（P＞0.05）.结论 渴络欣胶囊能明显减少早期糖尿病肾脏疾病患者NGAL、尿β2-MG水平,对早期DKD的肾小管功能有保护作用.     

黄芪菟箭合剂对糖尿病大鼠肾脏PI3K-Akt-mTOR信号通路的影响

目的:观察中药黄芪菟箭合剂对STZ诱导的糖尿病大鼠肾脏PI3K-Akt-m TOR信号通路的影响,并与雷帕霉素的作用进行比较。方法:糖尿病大鼠分为糖尿病对照组(DM组)、黄芪菟箭合剂治疗组(CM组)、雷帕霉素治疗组(RAPA组),并设正常大鼠对照组(CON组),分别给予不同治疗,12周后用ELISA法检测大鼠的尿白蛋白排泄率,免疫组化法观察肾小球ZO-1、nephrin蛋白表达;Western-blot法检测肾脏磷酸化Akt(Thr308)、磷酸化PTEN、磷酸化S6K的蛋白表达;应用qRT-PCR检测肾脏Akt、PTEN、S6K的mRNA表达。结果:CM组与RAPA组大鼠尿微量白蛋白排泄率均显著低于DM组,ZO-1、nephrin阳性表达显著高于DM组,且CM组与RAPA组之间无显著差别;CM组与RAPA组肾脏磷酸化S6K蛋白表达、S6KmRNA表达均显著低于DM组;CM组磷酸化PTEN蛋白及PTEN mRNA表达显著高于DM组,磷酸化Akt(Thr308)蛋白、Akt mRNA则低于DM组;但RAPA组磷酸化PTEN蛋白及PTEN mRNA表达以及磷酸化Akt(Thr308)蛋白、Akt mRNA表达与DM差异无统计学意义,RAPA组体重显著低于DM组及CM组、死亡率显著高于DM组及CM组。结论:雷帕霉素和黄芪菟箭合剂都可以有效地保护STZ诱导的糖尿病大鼠足细胞的完整性、减少尿微量白蛋白排泄率,但雷帕霉素存在显著毒副作用。     

川芎嗪联合坎地沙坦酯对高血压早期肾损害合并高尿酸血症的临床观察

目的 观察川芎嗪联合坎地沙坦酯对高血压早期肾损害合并高尿酸血症的影响.方法 将高血压早期肾损害合并高尿酸血症患者48例随机分为治疗组24例,对照组24例.治疗组用坎地沙坦酯8 mg联合川芎嗪160 mg加用生理盐水250ml静脉滴注;对照组仅用坎地沙坦酯8 mg.共观察4周.观察、记录治疗前、治疗后24 h尿微量白蛋白,血尿酸,尿素氮,血肌酐,胆固醇,甘油三酯,血压和药物不良反应.结果 2组均能降低患者尿微量白蛋白及血尿酸,但治疗组下降更加明显（P＜0.01）.结论 川芎嗪联合坎地沙坦酯治疗高血压早期肾损害合并高尿酸血症,能明显降低患者尿微量白蛋白、血尿酸,改善微循环,减轻肾损害,且无明显不良反应.     

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.     

氯沙坦对自发性2型糖尿病KKAy小鼠肾小球蛋白表达谱的影响

目的 利用比较蛋白质组学双向电泳技术研究体系来观察血管紧张素受体拮抗剂(ARB)氯沙坦对自发性2型糖尿病KKAy小鼠肾小球蛋白表达谱的影响.方法 8周龄自发性2型糖尿病KKAy小鼠随机分为氯沙坦治疗组(饮用水中喂入氯沙坦粉末10 mg+kg-1·d-1)和非治疗组;8周龄C57BL/6小鼠作为正常对照组.饲养12周后,经胸主动脉磁珠灌流分离肾小球,提取肾小球蛋白.DIGE最小荧光标记法标记,行二维荧光差异凝胶电泳.应用Typhoon多功能成像系统扫描凝胶,DeCyder 2D差异分析软件进行图像分析.采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定差异表达蛋白质.结果 KKAy小鼠的体质量、血糖和尿白蛋白排泄率较正常对照C57BL/6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小鼠尿白蛋白肌酐比[(539.71±100.23)mg/g比(728±177.19) mg/g]、肾小球基底膜增厚和系膜基质增生等肾脏病理损害,而对血糖无影响.通过DeCyder 2D差异分析软件,发现KKAy氯沙坦治疗组与KKAy非治疗组肾小球内表达差异大于1.1倍的蛋白质斑点62个.经肽质量指纹图分析,鉴定出41种蛋白质.其中表达上调的蛋白28种,包括甘油激酶、亚硫酸盐氧化酶、甘氨酸脒基转移酶、腺苷高半胱氨酸酶等;表达下调的蛋白质13种,包括3-巯基丙酮酸硫基转移酶、ATP合酶亚单位d、60 000热休克蛋白、线粒体应激蛋白70(又名75 000葡萄糖调节蛋白,GRP75)等.有6种蛋白在20周龄KKAy小鼠和C57BL/6小鼠肾小球内存在差异表达,氯沙坦治疗抑制了其在糖尿病状态下的表达变化,包括丙酮酸脱氢酶复合物的二氢硫辛酰赖氨酸残基乙酰基转移酶组分、琥珀酰辅酶A连接酶[GDP-形成]亚单位β、ATP合酶亚单位d、GRP75、核苷二磷酸盐结合部分X模体19和硒结合蛋白1.结论 氯沙坦可明显减轻KKAy小鼠尿白蛋白排泄率和肾脏病理损害;可抑制糖尿病状态下肾小球ATP合酶亚单位d、GRP75、硒结合蛋白1等蛋白的表达变化;可能通过减少线粒体活性氧簇产生,抑制氧化应激反应发挥其肾脏保护作用.     

Candesartan reduced advanced glycation end-products accumulation and diminished nitro-oxidative stress in type 2 diabetic KK/Ta mice.

BACKGROUND: Angiotensin-II induces nitro-oxidative stress in patients with diabetic nephropathy. Peroxynitrite and reactive oxide species can accelerate formation of advanced glycation end-products (AGEs). We investigated the effects of candesartan, an angiotensin-II type 1 receptor blocker (ARB), on the formation of AGEs and nitro-oxidative stress in type 2 diabetic KK/Ta mouse kidneys. METHODS: KK/Ta mice were divided into three treatment groups: an early treatment group receiving 4 mg/kg/day candesartan from 6 to 28 weeks of age, a late treatment group receiving the same candesartan dose from 12 to 28 weeks of age and a group receiving the vehicle for candesartan. BALB/c mice treated with vehicle were used as controls. We evaluated at 28 weeks the renal expressions of carboxymethyllysine, the receptor for AGE (RAGE), the p47phox component of NADPH oxidase, endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS) and 8-OHdG and nitrotyrosine by immunohistochemistry and/or by competitive RT-PCR. RESULTS: Kidneys from KK/Ta mice showed increased formation of AGEs, nitro-oxidative stress and RAGE expression and these were attenuated by candesartan treatment. Protein and mRNA expressions of p47phox and iNOS were upregulated in KK/Ta kidneys, which also showed increased immunostaining intensities of 8-OHdG and nitrotyrosine. Treatment with candesartan attenuated all of these changes and prevented significant albuminuria. There were no significant differences in the expression of eNOS among the four groups. CONCLUSIONS: These findings suggest that candesartan, an ARB, reduces AGE accumulation and subsequent albuminuria by down-regulating the NADPH oxidase p47phox component and iNOS expression and by attenuating RAGE expression in type 2 diabetic KK/Ta mouse kidneys.     

Effects of candesartan, an angiotensin II type 1 receptor blocker, on diabetic nephropathy in KK/Ta mice

BACKGROUND: Although therapeutic effects of angiotensin II type 1 receptor blocker (ARB) on renal injury in non-insulin dependant diabetes mellitus (NIDDM) have been demonstrated, the beneficial effects and their mechanisms in diabetic nephropathy have not been well evaluated. METHODS: KK/Ta mice were divided into three groups according to the treatment: candesartan 4 mg/kg/day from 6 to 28 weeks of age (group I; early treatment); from 12 to 28 weeks of age (group II; late treatment); only vehicle (group III). BALB/c mice treated with vehicle were used as controls (group IV). Body weight (BW), systolic blood pressure (SBP), blood glucose, urinary type IV collagen and albumin excretion were measured every 4 weeks. Morphometry and immunohistology of albumin, transforming growth factor-beta1 (TGF-beta1) and Smad7 were performed in all groups. RESULTS: BW and blood glucose were higher in groups I, II and III than in group IV from 8 weeks. SBP was markedly reduced in groups I and II compared with group III (p < 0.05, p < 0.005). Urinary type IV collagen and albumin excretion were increased in group III compared to group IV (p < 0.05, p < 0.005), whereas they were reduced in groups I and II when compared to group III (p < 0.05). Morphometric analysis revealed that the whole glomerular area (WGA), glomerular tuft area (GTA), extracellular matrix area (ECMA) and intraglomerular cell nuclei number (NIGCN) were significantly reduced in groups I, II and IV compared to group III at 28 weeks. In immunohistochemistry, TGF-beta1 expression in both glomeruli and tubules of groups I and II decreased compared to that of group III at 28 weeks, while Smad7 in group III glomeruli was reduced compared to that in groups I and II. CONCLUSIONS: It appears that candesartan reduced urinary type IV collagen and albumin excretion, and attenuated glomerular hypertrophy and mesangial matrix accumulation by the TGF-betaS/Smad signaling pathway in KK/Ta mice with diabetic nephropathy.     

Identification of epistatic interaction involved in obesity using the KK/Ta mouse as a Type 2 diabetes model: is Zn-alpha2 glycoprotein-1 a candidate gene for obesity?

The KK/Ta strain serves as a suitable polygenic mouse model for the common form of type 2 diabetes associated with obesity in humans. Recently, we reported the susceptibility loci contributing to type 2 diabetes and related phenotypes in KK/Ta mice. In this study, we focused on expression in the kidneys and liver of KK/Ta and BALB/c mice using differential display (DD) PCR. Zn-alpha(2) glycoprotein-1 (Azgp1) mRNA levels were increased in the kidneys and liver in KK/Ta mice, and sequence analysis revealed a missense mutation. We analyzed the relationship between this polymorphism and various phenotypes in 208 KK/Ta x (BALB/c x KK/Ta) F1 backcross mice. Statistical analysis revealed that Azgp1 and D17Mit218 exhibit a suggestive linkage to body weight (8 weeks) (logarithm of odds 2.3 and 2.9, respectively). Moderate gene-gene interactions were observed at these loci. Adiponectin mRNA levels in 3T3-L1 cells transfected with the expression pcDNA 3.1 vector containing Azgp1 coding sequence of KK/Ta mice were significantly higher than those of BALB/c mice. These results suggest that Azgp1 is a possible candidate gene for regulation of body weight, elucidation of polygenic inheritance, and age-dependent changes in the genetic control of obesity.     

Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB.

BACKGROUND: Angiotensin II (Ang II) can up-regulate nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase, whose product superoxide anion (O2-) can interact with nitric oxide (NO) to form peroxynitrite (ONOO-). We tested the hypothesis that Ang II subtype 1 (AT1) receptor activation enhances oxidative stress and nitrotyrosine deposition in the kidneys of rats with diabetes mellitus (DM). METHODS: After two weeks of streptozotocin-induced DM, rats received either no treatment, an angiotensin-converting enzyme inhibitor (ACEI) or an angiotensin receptor blocker (ARB) for two weeks. At four weeks, renal expression of the p47phox component of NAD(P)H oxidase, endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and nitrotyrosine were evaluated by Western blot and immunohistochemistry and related to plasma lipid peroxidation products (LPO), hydrogen peroxide production in the kidney and 24-hour protein excretion. RESULTS: Immunoreactive expression of p47phox and eNOS were increased in DM with an increase in plasma LPO, renal hydrogen peroxide production and nitrotyrosine deposition. Expression of nNOS was unaltered. Treatment with either ACEI or ARB prevented all these findings and also prevented significant microalbuminuria. The treatments did not affect the elevated blood sugar, nor did DM or its treatment affect the blood pressure or the creatinine clearance. CONCLUSION: Early proteinuric diabetic nephropathy increases renal expression of the p47phox component of NAD(P)H oxidase and eNOS with increased indices of systemic and renal oxidative/nitrosative stress. An ACEI or an ARB prevents these changes and prevents the development of proteinuria, independent of blood pressure or blood sugar. This finding indicates a pathogenic role for AT1 receptors in the development of oxidative damage in the kidneys during early DM.     

Chromosomal mapping of a quantitative trait locus for the development of albuminuria in diabetic KK/Ta mice.

BACKGROUND: The KK/Ta mouse strain serves as a suitable polygenic model for human type 2 diabetes. We previously reported a genome-wide linkage analysis of KK/Ta alleles contributing to type 2 diabetes and related phenotypes such as fasting hyperglycaemia, glucose intolerance, hyperinsulinaemia, obesity and dyslipidaemia. METHODS: Since KK/Ta mice spontaneously develop renal lesions closely resembling those in human diabetic nephropathy, we investigated the susceptibility loci using the KK/Ta x (BALB/c x KK/Ta) F1 backcross progeny in the present study. RESULTS: A genome-wide analysis of susceptibility loci for albuminuria with microsatellite-based chromosomal maps showed a contributing KK/Ta locus, provisionally designated UA-1, with a significant linkage with the interval on chromosome 2 at 83.0 cM close to the microsatellite marker D2Mit311 with a maximum LOD of 3.5 (chi(2) = 13.2, P = 0.0003). UA-1 was different from the susceptibility loci contributing to type 2 diabetes, which we earlier identified. The mode of inheritance differed from that of hypertension. The progeny homozygous for UA-1 showed significantly higher urinary albumin levels. CONCLUSIONS: Although there were no significant correlations between urinary albumin levels and other diabetic phenotypes, the group of progeny homozygous for both UA-1 and alleles for fasting hyperglycaemia showed the highest urinary albumin levels. Thus, UA-1 appears to increase the risk of diabetic nephropathy, particularly in individuals susceptible to fasting hyperglycaemia, in a gene dosage-dependent manner. There are potentially important candidate genes that may be relevant to diabetic nephropathy.     

Chronic diabetic nephropathy: role of inducible nitric oxide synthase

Nitric oxide (NO) is a multifunctional mediator that has been implicated in the short-term hemodynamic alterations that occur in acute streptozocin (STZ)-induced diabetes. We investigated the role of NO produced by inducible nitric oxide synthase (iNOS) in chronic STZ diabetic nephropathy. Diabetes was induced in C57BL/6 and iNOS knockout (KO) mice with two intraperitoneal injections of STZ, 100 mg/kg. Animals were maintained without insulin treatment for 40 weeks. There were no significant differences between the strains in blood urea nitrogen (BUN), serum creatinine or glucose concentration, or urinary protein excretion during the entire observation period. Urinary nitrite + nitrate excretion was significantly lower in iNOS KO mice compared to control animals at all time points; in C57 mice, urinary nitrite declined progressively with more prolonged duration of diabetes. Renal hypertrophy (kidney weight/body weight) was noted in both strains of mice. However, histopathological assessment of renal tissue specimens at 16 and 40 weeks demonstrated increased mesangial hypercellularity and expansion as well as more prominent tubulointerstitial fibrosis in iNOS KO versus C57 mice. These changes were accompanied by increased interstitial deposition of type I collagen at 16 and 40 weeks in iNOS KO mice. Glomerular basement membrane staining for type IV collagen was also increased at 40 weeks in diabetic iNOS KO mice. While iNOS protein was undetectable in any of the kidney specimens obtained from either strain, eNOS was present throughout the course of chronic STZ diabetes. Moreover, eNOS expression was significantly increased by approximately 40% at 16 and 40 weeks of observation in iNOS KO versus C57 mice. There was no difference in renal cortical malondialdehyde content between the strains early or late in the disease course. In time control animals, there was no evidence of renal histopathological damage in iNOS KO or C57 mice after 40 weeks. We conclude that iNOS-derived NO modulates glomerulosclerosis and tubulointerstitial fibrosis in chronic STZ nephropathy. This action is probably a result of the direct actions of NO on the synthesis and degradation of extracellular matrix proteins. Received: 28 February 2001 / Revised: 10 August 2001 / Accepted: 13 August 2001