Oxidative stress, lipid hyperoxides and diabetic nephropathy

氧化应激、脂质过氧化物和糖尿病肾病

• 批准号: 15590931
• 负责人: TSUKAMOTO Kazuhisa
• 金额: $ 2.24万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590931/
• 关键词: Diabetes Mellitus; Diabetic Nephropathy; Oxidative stress; Lipid hyperoxide; Gene transfer; PAF acetylhydrolase; Glomerulosclerosis

Several in vitro studies have clarified that lipid hyperoxides and oxidative stress play a crucial role in the progression of diabetic nephropathy. However, no study has demonstrated the role of lipid hyperoxides or oxidative stress in the progression of diabetic nephropathy in vivo. In order to elucidate whether the amelioration of oxidative stress would lead to the inhibition of progression of diabetic nephropathy, we designed the experiment to overexpress PAF acetylhydrolase (PAFAH), an enzyme which inactivates lipid hyperoxides and ameliorate oxidative stress, in the animal models of diabetic nephropathy utilizing adenovirus-mediated gene transfer. In addition, in this research period, we examined precisely the mechanism of the amelioration of glomerulosclerosis caused by the overexpression of PAFAH in Imai rat, an animal model of glomerulosclerosis.First, we examined db/db mice, which has previously reported to develop diabetic nephropathy (JCI 95 : 2338, 1995). With the administr … More ation of AdPAFAH, an adenoviral vector which encodes human PAFAH, the plasma PAFAH activity increased to 42 folds. However, contrary to the previous reports, the protein uria did decrease even in the control mice, and we resigned this animal model. Next, we tried to utilize SHR/NDmc-cp (fat/fat) rat, which is also reported to develop diabetic nephropathy (J Am Soc Nephrol 14 : 1212, 2003). However, this animal model did not show hyperglycemia even if it was given high sucrose containing diet ; thus we resigned this animal model. Then we moved to the next model which is reported to develop diabetic nephropathy in 2000 (Eur J Pharmacol 398 : 381, 2000). Administration of streptozosin (STZ) 200mg/kg to C57BL/6 mice developed diabetes and diabetic nephropathy. In addition, we could observe a tendency in the amelioration of protein uria by the overexpression of PAFAH. However, at the same time, we found that administration of STZ 200 mg/kg caused severe diabetes in mice, resulting in the death of the animals in the experimental periods. Thus we examined the appropriate dose of STZ, and found that 140 mg/kg would be the best dose for the experiment. We are now analyzing the effect of overexpression of PAFAH in this model.We have previously observed that in Imai rat, an animal model of glomerulosclerosis, administration of AdPAFAH and overexpression of PAFAH resulted in the amelioration of glomerulosclerosis judging from proteinuria and histological examination. In addition, we have observed that PAFAH protein was found exclusively on mesangial cells in the glomeruli. Thus we examined the mechanism of this amelioration precisely during this research period. In situ hybridization analysis revealed that the PAFAH protein is not expressed in the glomeruli after adenovirus-mediated gene transfer. Administration of HDL rich in PAFAH to Imai rats resulted in the deposition of PAFAH protein in the glomeruli. Furthermore, immunostaining of HNE, which is a marker for oxidative stress, revealed the amelioration of oxidative stress in the glomeruli in the PAFAH overexpressing rat. We did not observe any changes in the plasma isoprostane levels. Thus we could confirm that the PAFAH protein overexpressed in the liver delivered to glomeruli through HDL, reduced oxidative stress locally, and ameliorates glomerulosclerosis in Imai rats. Less

几项体外研究阐明了脂质高氧化物和氧化应激在糖尿病肾病的进展中起着至关重要的作用。然而，尚无研究表明脂质高氧或氧化应激在体内糖尿病性肾病进展中的作用。为了阐明氧化应激的改善是否会导致糖尿病性肾病的进展，我们设计了该实验以过表达PAF PAF乙酰水合酶（PAFAH），一种使脂质高氧化物和氧化氧化物的氧化型转移的酶的酶抑制了脂质高氧化物的氧化酶，该酶使糖尿病模型融合了依赖型肾上腺素。此外，在这一研究期间，我们精确地检查了由Pafah在Imai大鼠中的过度表达引起的肾小球硬化的机制，Imai大鼠是肾小球硬化的动物模型。随着行政局……更多地编码人类Pafah的腺病毒矢量Adpafah，等离子体Pafah活动增加到42倍。但是，与先前的报道形成鲜明对比的是，即使在对照小鼠中，蛋白质乌里亚也确实减少了，我们辞去了这种动物模型。接下来，我们尝试利用SHR/NDMC-CP（脂肪/脂肪）大鼠，也据报道会发展糖尿病性肾病（J Am Soc Soc Nephrol 14：1212，2003）。但是，即使给予含饮食的高蔗糖，这种动物模型也没有表现出高血糖。因此，我们辞去了这种动物模型。然后，我们搬到了下一个模型，该模型据报道在2000年发展为糖尿病性肾病（Eur J Pharmacol 398：381，2000）。对C57BL/6小鼠的链托辛（STZ）200mg/kg的施用产生了糖尿病和糖尿病性肾病。此外，我们可以通过Pafah的过表达观察到蛋白质URIA的改善趋势。但是，与此同时，我们发现STZ的给药200 mg/kg在小鼠中引起严重的糖尿病，导致动物在实验期间死亡。我们检查了适当的STZ剂量，并发现140 mg/kg将是实验的最佳剂量。现在，我们在该模型中分析了Pafah过度表达的作用。我们先前观察到，在Imai大鼠中，肾小球硬化的动物模型，ADPAFAH的给药和PAFAH的过表达导致从蛋白尿和蛋白尿和组织学检查中判断出肾小球硬化的层状。此外，我们已经观察到Pafah蛋白仅在肾小球中的弥赛亚细胞上发现。在这一研究期间，我们精确地检查了这种麻醉症的机制。原位杂交分析表明，在腺病毒介导的基因转移后，Pafah蛋白在肾小球中未表达。富含Pafah的HDL给IMAI大鼠的给药导致Pafah蛋白在Glomerulli中的沉积。此外，HNE的免疫染色是氧化应激的标志物，揭示了Pafah过表达大鼠肾小球中氧化应激的改善。我们没有观察到血浆异端水平的任何变化。我们可以证实，Pafah蛋白在肝脏中通过HDL递送至肾小球，局部减少氧化应激，并改善IMAI大鼠的肾小球硬化。较少的

项目成果

Noto H, Tsukamoto K, et al.: "Modulation of HDL metabolism by probucol in complete cholesteryl ester transfer protein deficiency"Atherosclerosis. 171. 131-136 (2003)

Noto H、Tsukamoto K 等人：“普罗布考在完全胆固醇酯转移蛋白缺乏症中调节 HDL 代谢”动脉粥样硬化。

Shintani Y, Tsukamoto K, et al.: "Hepatitis C Virus Infection and Diabetes : Direct Involvement of the Virus in the Development of Insulin Resistance"Gastroenterology. 126. 840-848 (2004)

Shintani Y、Tsukamoto K 等人：“丙型肝炎病毒感染和糖尿病：病毒直接参与胰岛素抵抗的发展”胃肠病学。

Ishizaka N, Tsukamoto K, et al.: "Association between insulin resistance and carotid arteriosclerosis in subjects with normal fasting glucose and normal glucose tolerance"Arteriosclerosis Thrombosis and Vascular Biology. 23. 295-301 (2003)

Ishizaka N、Tsukamoto K 等人：“空腹血糖和糖耐量正常的受试者中胰岛素抵抗与颈动脉硬化之间的关联”动脉硬化血栓形成和血管生物学。

Noto H, Tsukamoto K, et al.: "Human plasma platelet activating factor-acetylhydrolase binds to all the murine lipoproteins, conferring protection against oxidative stress"Arteriosclerosis Thrombosis and Vascular Biology. 23. 829-835 (2003)

Noto H、Tsukamoto K 等人：“人血浆血小板激活因子乙酰水解酶与所有鼠类脂蛋白结合，提供针对氧化应激的保护”动脉硬化血栓形成和血管生物学。

Hara M, Tsukamoto K, et al.: "Isoform-dependent cholesterol efflux from macrophages by apolipoprotein E is modulated by cell surface proteoglycans"Arteriosclerosis Thrombosis and Vascular Biology. 23. 269-274 (2003)

Hara M、Tsukamoto K 等人：“载脂蛋白 E 从巨噬细胞中流出的异构体依赖性胆固醇受到细胞表面蛋白聚糖的调节”《动脉硬化血栓形成和血管生物学》。