Oxidative stress, lipid hyperoxides and diabetic nephropathy

几项体外研究表明，脂质过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，没有研究证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抑制糖尿病肾病的进展，我们设计了一项实验，利用腺病毒介导的基因转移，在糖尿病肾病动物模型中过表达PAF乙酰水解酶（PAFAH），该酶能灭活脂质过氧化物并改善氧化应激。此外，在本研究期间，我们在肾小球硬化动物模型Imai大鼠中精确检测了PAFAH过表达对肾小球硬化改善的机制。首先，我们研究了db/db小鼠，这些小鼠以前曾报道发生糖尿病肾病（JCI 95: 2338, 1995）。AdPAFAH是一种编码人PAFAH的腺病毒载体，随着AdPAFAH剂量的增加，血浆PAFAH活性增加到42倍。然而，与之前的报道相反，即使在对照组小鼠中，蛋白尿也确实减少了，因此我们放弃了这个动物模型。接下来，我们尝试使用SHR/NDmc-cp（脂肪/脂肪）大鼠，这也有报道发生糖尿病肾病（J Am Soc Nephrol 14: 1212,2003）。然而，即使给予高蔗糖饮食，该动物模型也没有表现出高血糖；因此，我们放弃了这个动物模型。然后我们转移到2000年报道的下一个模型糖尿病肾病（Eur J Pharmacol 398: 381,2000）。链脲菌素（STZ） 200mg/kg给药C57BL/6小鼠发生糖尿病和糖尿病肾病。此外，我们可以观察到PAFAH的过表达有改善蛋白尿的趋势。但与此同时，我们发现给药STZ 200 mg/kg会引起小鼠严重的糖尿病，导致实验期间动物死亡。因此，我们考察了STZ的适宜剂量，发现140 mg/kg是本实验的最佳剂量。我们现在正在分析PAFAH过表达对该模型的影响。我们之前在肾小球硬化动物模型Imai大鼠中观察到，从蛋白尿和组织学检查来看，给药AdPAFAH和过表达PAFAH可改善肾小球硬化。此外，我们观察到PAFAH蛋白只存在于肾小球的系膜细胞上。因此，我们在研究期间精确地考察了这种改善的机制。原位杂交分析显示，经腺病毒介导的基因转移后，PAFAH蛋白在肾小球中不表达。给Imai大鼠注射富含PAFAH的HDL可导致肾小球内PAFAH蛋白沉积。此外，氧化应激标志物HNE的免疫染色显示PAFAH过表达大鼠肾小球氧化应激的改善。我们没有观察到血浆异前列腺素水平的任何变化。因此，我们可以证实PAFAH蛋白在肝脏中过表达通过HDL传递到肾小球，局部减少氧化应激，改善Imai大鼠肾小球硬化。少

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