| Co-Investigator(Kenkyū-buntansha) |
MAE Hiromu Hyogo College of Medicine, Department of Pediatrics, Research Associate, 医学部, 助手 (50309457)
OOSHIMA Keisuke Hyogo College of Medicine, Department of Pediatrics, Research Associate, 医学部, 助手 (90268540)
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| Research Abstract |
Glomerular sclerosis and tubulo-interstitial fibrosis are the common cause of end-stage renal failure and are supposed to be irreversible, regardless of the primary etiology of renal disease. Hepatocyte growth factor (HGF) is now well known as a cytokine with numerous functions on diverse cell survival by inhibiting apoptosis and tissue regeneration. Compared with repeatedly injection of HGF protein, gene therapy is the best for its efficiency and financial economy. In this study, we studied the effects and possible mechanisms of HGF gene therapy on tubular cell survival and blockage of renal fibrosis in chronic obstructed nephropathy by unilateral ureteral obstruction (UUO).
An in vivo transfection procedure of repeatedly transducing skeletal muscles with HGF gene using liposomes containing the hemagglutinating virus of Japan (HVJ liposome) was tested on UUO rats. Thirty 5-week-old male Sprague-Dawley rats were subjected to complete UUO or Sham operation. UUO rats were randomly separat
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ed into two groups. The treated group received repetitive intramuscular injection of HVJ liposomes containing 800μg pUC-SRα/HGF once a week, while the untreated group was injected with BSS. Then kidney tissues were collected for further assay at 2w or 4w after operations. Expression of HGF and c-Met were examined by in situ hybridization. Interstitial fibrosis and macrophage infiltration were evaluated by Masson's Trichrome staining, α-SMA and ED-1 immunostaining. Cell survival indices including PCNA, Bcl-2, Bcl-xL and Bax were measured by immunohistochemistry and western blots. Apoptosis was determined by TUNEL method.
After HGF-HVJ-liposome gene transfer, endogenous HGF and c-Met were up regulated. Renal interstitial volume and fibroblast activity were suppressed both at day 14 and 28 after UUO (p<0.05 or 0.01). The tubular macrophage infiltration in the late-stage obstructed kidneys was significantly reduced. Tubular atrophy progression after the obstruction was also suppressed. Tubular cell proliferation was activated while apoptosis was inhibited, especially at the late-stage of UUO. Bcl-2 was enhanced in HGF transfected UUO rats, while no changes of Bcl-xL and Bax were found. Less
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