NOIRI Eisei The University of Tokyo, Faculty of Medicine, Assistant, 医学部附属病院, 助手 (00301820)
TOGO Masako The University of Tokyo, Faculty of Medicine, Medical Staff, 医学部附属病院, 医員
HARA Masumi The University of Tokyo, Faculty of Medicine, Medical Staff, 医学部附属病院, 医員
ISO-O Naoyuki The University of Tokyo, Faculty of Medicine, Medical Staff, 医学部附属病院, 医員
|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2004 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 2003 : ¥2,500,000 (Direct Cost : ¥2,500,000)
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
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