|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1999 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1998 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Recently, we have found that gene expression of peritoneal aquaporins (AQP1 and 4) was enhanced transitorily at the initial phase of continuous peritoneal dialysis (PD) in rats. To study the involvement of AQP in the ulltrafiltration loss occurred in long term CAPD patients, we designed long term experimental PD for 2 months in rats. Small catheter was settled in the abdominal cavity for exchanging dialysate and sampling.. Physiological saline (for control) or glucose-containing human-use dialysate(1.35% or 4.0%) were administered and exchanged twice a day. Daily monitored effluent volume (ultrafiltration volume) was increased in the initial phase and subsequently fixed, as observed in the previous experiment. Histological changes in the peritoneum such as mesothelium detachment or submesothelial fibrosis were not obvious. The effluent volume was gradually decreased after 5 weeks although we failed to obtain the statistical significance. Gene expression of peritoneal AQP1 and 4 after 8
weeks dialysis was inclined to be decreased comparing to that before dialysis (statistically insignificant) studied by RT-PCR combined with ELISA analysis. In situ hybridization study revealed that the signal accumulation in the capillary endothelium was less obvious after 8 weeks dialysis,but it was not significant. To confirm the involvement of AQP in the ultrafiltration failure in patients with long-term CAPD therapy, further study must be required. Next, to study the involvement of mechanical stretch in the induction of AQP gene expression, we designed in vivo and in vitro experiments. For in vivo experiment, small catheter with balloon was settled into the rats abdominal cavity to expand the peritoneum without dialysis. For in vitro experiment, mesothelial cells obtained by mechanical scratch of the peritoneum were cultured on the rubber plate, for subsequent application to 30% statistic stretch or 60 Hz cycled stretch. Peritoneum expansion in vivo generated transient enhancement of AQP gene expression at the day 3, which was similar pattern to that in experimental dialysis. However, there was no significant difference in in vitro experiement, suggesting that indirect effect of peritoneal expansion might be involved in the transient enhancement of AQP gene expression. Clinically, unexpected recovery from ultrafiltration failure in CAPD patients is sometimes experienced after few weeks discontinuation of PD. The peritoneal expansion induced AQP gene expression may be involved in the phenomenone.
一方短期実験で得られた一過性のAQP遺伝子発現増加への物理的進展刺激の関与を考え、in vivo 実験としてラット腹腔内にバルーン付きカテーテルを留置、10-15mlの生理食塩水を注入し、経時的にmRNA発現量の検討を行った。またin vitro実験としてラット壁側腹膜よりコラゲネース処理で得られた中皮細胞をシリコン製培養容器上で培養し、30%の静的伸展刺激及び60Hzの伸縮刺激を負荷し、経時的にmRNA発現量の検討を行った。In vivo実験ではday3をピークとする一過性の発現量が見られ、透析によらない腹膜の進展でもAQP遺伝子発現が誘導されることが確認された。しかしin vitro実験では明らかな発現変化は確認できなかった。以上の結果より長期PD患者に見られる限外濾過不全にAQP遺伝子発現が関与する可能性が示唆されたものの、なお長期間での検討が必要と思われた。一方短期的なAQP遺伝子発現増加には間接的な腹膜進展刺激が関与すると思われ、臨床的に観察される一時的なPD休止後の限外濾過量増加を反映するものである可能性が考えられた。 Less