Genetic modification of cold-preserved renal grafts using HSP70 or bcl-2 HVJ-liposome method.
BACKGROUND
We tested the hypothesis that the best time for genetic modification is while the cell viability of the graft is reduced for long-term preservation. The hemagglutinating virus of Japan (HVJ)-liposome method, a nonviral gene transfer technique, was used with a luciferase gene to test the efficacy of protein induction under the critical preservation time. Furthermore, we tested this genetic modification with heat shock protein (HSP) 70 or bcl-2 genes to prevent primary nonfunction (PNF) after long-term preservation.


METHODS
Orthotopic rat renal transplantation (RT) was performed using the cuff technique in the syngeneic combination of Lew (major histocompatible complex, haplotype: RT1(l)). Rat kidney grafts were preserved for 24 or 48 h in University of Wisconsin (UW) or Ringer's lactate solution using HVJ method with the luciferase gene. Rats with gene-transfected kidneys were re-laparotomized 48 h after transplantation to estimate the lack of arterial flow in the graft and killed for histological evaluation of the degree of PNF luciferase intensity assay. Then, two functional genes (HSP70 or bcl-2) were tested for the occurrence of PNF and histological and immunohistochemical analysis of the grafted kidneys preserved for 48 h in the UW solution.


RESULTS
In the kidneys preserved for 24 h, 50% of the Ringer's lactate group had PNF; but all of the UW group had sufficient blood flow. The graft viability was well corrected by the degree of luciferase intensity. The PNF rate was significantly suppressed in the bcl-2 gene-transfer group, and tended to be reduced in the HSP70 group.


CONCLUSIONS
The HVJ-liposome method effectively induced the foreign gene for kidney grafts even in the cold-preservation solution. Induction of bcl-2 or the HSP70 gene reduced the occurrence of PNF in the rat renal graft. The results suggest that gene transfer not only maintains graft viability, but also graft activation.
Kita
Junji
J
Second Department of Surgery, Dokkyo University School of Medicine, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan.
Kobayashi
Eiji
E
Hishinuma
Akira
A
Kaneda
Yasufumi
Y
D000818:Animals; D015925:Cryopreservation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D016030:Kidney Transplantation; D008081:Liposomes / Q000378:metabolism*; D008297:Male; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew
D018840:HSP70 Heat-Shock Proteins; D008081:Liposomes; D019253:Proto-Oncogene Proteins c-bcl-2