Oxidative stress, lipid hyperoxides and diabetic nephropathy

一些体外研究已经阐明，过氧化脂质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，还没有研究表明脂质过氧化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，我们设计了在腺病毒介导的糖尿病肾病动物模型中过度表达PAF乙酰水解酶(PAFAH)的实验。PAFAH是一种抑制脂质过氧化和改善氧化应激的酶。此外，在本研究期间，我们精确地研究了PAFAH在肾小球硬化动物模型Imai大鼠中过度表达导致肾小球硬化改善的机制。首先，我们研究了db/db小鼠，这是先前报道的糖尿病肾病(JCI 95：2338,1995)。使用管理员…在编码人PAFAH的腺病毒载体AdPAFAH作用下，血浆PAFAH活性增加了42倍。然而，与之前的报道相反，即使在对照组小鼠中，URIA蛋白也确实减少了，我们放弃了这个动物模型。接下来，我们试图利用SHR/NDMC-cp(FAT/FAT)大鼠，它也被报道患有糖尿病肾病(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，我们放弃了该动物模型。然后我们转到下一个模型，据报道在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C57BL/6小鼠造成糖尿病和糖尿病肾病。此外，我们还可以观察到PAFAH过表达对URIA蛋白的改善趋势。然而，我们同时发现，给小鼠注射链脲佐菌素200 mg/kg会引起严重的糖尿病，导致实验期动物死亡。因此，我们研究了STZ的合适剂量，发现140 mg/kg是实验的最佳剂量。我们现在正在分析PAFAH的过度表达在这个模型中的作用。我们以前在IMAi大鼠的肾小球硬化动物模型中观察到，从蛋白尿和组织学检查来看，AdPAFAH的应用和PAFAH的过度表达导致肾小球硬化的改善。此外，我们还观察到PAFAH蛋白仅存在于肾小球系膜细胞中。因此，在本研究期间，我们准确地考察了这种改善的机制。原位杂交分析表明，腺病毒介导的基因转移后，PAFAH蛋白在肾小球中不表达。IMAI大鼠给予富含高密度脂蛋白的PAFAH后，肾小球中PAFAH蛋白沉积。此外，作为氧化应激标志的HNE的免疫染色显示，PAFAH过度表达的大鼠肾小球内的氧化应激有所改善。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，通过高密度脂蛋白传递到肾小球，减轻局部氧化应激，改善IMAi大鼠肾小球硬化。较少

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