| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Kidney-targeted gene transfer has the potential to be one of the most important tools for broadening our understanding of renal disease processes and revolutionizing the treatment of renal diseases. Previous gene-transfer methods using nonviral vectors administered via renal arterial, pelvic, or ureteric routes into the glomerulus, tubules, or interstitial fibroblasts have shown low-level expression for < 1 month. The peritubular capillaries (PTC) network is one of the main targets of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. We believe the interstitium containing the PTC is an important candidate site for gene transfer. To access the PTC, we retrogradely injected a lacZ expression plasmid in Ringer's solution into the renal vein of rats. We found lacZ expression only in the interstitial fibroblasts near the PTC of the injected kidney by immunoelectron microscopic analysis. Nephrotoxicity due to gene trans
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fer was not apparent. We used a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo, in a reporter gene. We found maximal Epo expression when the DNA solution was injected within 5 sec, and with a volume of 1.0 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 μg. We detected the transgene-derived Epo mRNA by RT-PCR only in the kidneys injected with pCAGGS-Epo. After an injection of 100 μg of pCAGGS-Epo, the serum Epo levels peaked at 208.3 ± 71.8 mU/ml at week 5, and gradually decreased to 116.2 ± 38.7 mU/ml at week 24. A similar pattern was seen using smaller doses of plasmid, 2 or 30 μg of pCAGGS-Epo. Transgene-derived Epo secretion caused significant erythropoiesis. Next, we tested whether rat kidney could be targeted under the clamping renal vein only. We obtained maximal Epo expression when the DNA solution was injected within 2 sec, and with a volume of 0.5 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 μg. After an injection of 100 μg of pCAGGS-Epo, the serum Epo levels peaked at 423.8 ± 320.1 mU/ml at week 3, and gradually decreased to 161.5 ± 125.2 mU/ml at week 24. Transgene-derived Epo secretion gave significant erythropoiesis. The novel technique is simple, safe, and allows high-level, long-term stable gene expression and specific gene transfer to the fibroblasts near the PTC, which makes it particularly appealing for potential for future applications in humans. Less
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