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Oxidative stress, lipid hyperoxides and diabetic nephropathy

氧化应激、脂质过氧化物和糖尿病肾病

基本信息

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

项目摘要

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

一些体外研究已经阐明脂质过氧化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，没有研究表明脂质过氧化物或氧化应激在体内糖尿病肾病进展中的作用。为了阐明氧化应激的改善是否会导致糖尿病肾病进展的抑制，我们设计了实验，在糖尿病肾病动物模型中过表达PAF乙酰水解酶（PAFAH），一种灭活脂质过氧化物并改善氧化应激的酶，利用腺病毒介导的基因转移。此外，在本研究期间，我们在肾小球硬化的动物模型Imai大鼠中精确地研究了由PAFAH过表达引起的肾小球硬化改善的机制。首先，我们研究了db/db小鼠，其先前已报道发生糖尿病肾病（JCI 95：2338，1995）。与行政人员 ...更多信息重组腺病毒AdPAFAH后，血浆PAFAH活性提高42倍。然而，与以前的报道相反，即使在对照组小鼠中，蛋白尿也确实减少，我们放弃了这种动物模型。接下来，我们尝试利用SHR/NDmc-cp（脂肪/脂肪）大鼠，其也被报道发展为糖尿病肾病（J Am Soc Nephrol 14：1212，2003）。然而，即使给予高蔗糖饮食，该动物模型也没有显示高血糖症;因此，我们放弃了该动物模型。然后，我们转移到下一个模型，据报道其在2000年发展为糖尿病肾病（Eur J Pharmacol 398：381，2000）。链脲佐菌素（STZ）200 mg/kg给C57 BL/6小鼠灌胃，诱发糖尿病和糖尿病肾病。此外，我们可以观察到PAFAH过表达改善蛋白尿的趋势。然而，同时，我们发现STZ 200 mg/kg给药引起小鼠严重的糖尿病，导致动物在实验期间死亡。因此，我们对STZ的适宜剂量进行了研究，发现140 mg/kg是本实验的最佳剂量。我们正在分析PAFAH过表达在该模型中的作用。我们先前观察到，在肾小球硬化的动物模型Imai大鼠中，给予AdPAFAH和PAFAH过表达导致从蛋白尿和组织学检查判断的肾小球硬化的改善。此外，我们还观察到PAFAH蛋白只存在于肾小球的系膜细胞上。因此，我们在此研究期间，正是这种改善的机制。原位杂交分析显示，PAFAH蛋白在腺病毒介导的基因转移后的肾小球中不表达。富含PAFAH的HDL对Imai大鼠的给药导致PAFAH蛋白在肾小球中的沉积。此外，HNE的免疫染色，这是一个氧化应激的标志物，揭示了PAFAH过表达大鼠肾小球中的氧化应激的改善。我们未观察到血浆异前列烷水平的任何变化。因此，我们可以证实，在肝脏中过表达的PAFAH蛋白通过HDL递送到肾小球，局部降低氧化应激，并改善Imai大鼠的肾小球硬化。少