腹膜硬化症は腹膜透析の重大な合併症であり,誘因の一つとして長期にわたる腹膜への高糖暴露が報告されているが,その発症機序に関しては不明な点が多い.腹膜中皮細胞は透析液による暴露を最も受ける細胞である.今回我々は,培養腹膜中皮細胞に高糖刺激を行い, mitogen-activated protein kinase (MAPK)の動態を評価した.ラット腹膜中皮細胞は 0.5% FCS含有DMEM培地で24時間培養した後, 4%グルコースと4%マニトール刺激を行った.細胞増殖能はWST-1法を用いて測定し,上清中のfibronectin (FN)およびMAPKの変化はWestern blottingで半定量化した. FN mRNAの変化はRT-PCRで判定した. 4%グルコースと4%マニトール刺激は,いずれも経時的にFN mRNAとFN蛋白を増加させた.また, 4%グルコースと4%マニトール刺激は,いずれもMAPKのうちextracellular signal-regulated kinase (ERK)とp38MAPKを活性化し, MAPK阻害剤で4%グルコースによるFN mRNA増加は抑制された.最後に, 4%グルコース刺激によるFNの増加は, transforming growth factor (TGF)-β中和抗体の添加で抑制されず,培養上清中へのTGF-βの分泌増加を伴わなかった.これらの結果より,高糖刺激によるFNの増加には, TGF-βを介さないMAPKの活性化が関与していると考えられた.同濃度のマニトール刺激でも,グルコースと同様に MAPKの活性化やFNの増加が見られたことから,高糖刺激は浸透圧刺激として作用したものと思われた.したがって,高糖刺激によりMAPKが活性化された結果として,腹膜中皮細胞によるFNの産生が亢進し,腹膜線維化を惹起していることが示唆された.Peritoneal fibrosis is a serious complication in long-term continuous ambulatory peritoneal dialysis (CAPD) patients, but the underlying mechanism is not well understood. Since high glucose activates the p38 mitogen-activated protein kinase (MAPK) pathway in various kinds of cells, and because mesothelial cells always exposed to high glucose dialysate, we investigated the involvement of MAPK in rat peritoneal mesothelial cells (PMCs) under high glucose conditions. Rat PMCs were grown in Dulbecco\u27s modified Eagle\u27s medium with 0.5% FBS in 24 hours and then exposed to 4% glucose or 4% mannitol. Cell viability was assessed by using WST-1 assay. Fibronectin (FN) accumulation in the supernatant was determined by Western blot. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to determine mRNA and protein expression, respectively. High glucose and mannitol caused time-dependently increase in FN accumulation and FN mRNA in rat PMCs. High glucose also activated the extracellulaer signal-regulated kinase (ERK) and p38 MAPK. These increase in FN accumulation were not inhibited with anti-transforming growth factor (TGF)-β neutralizing antibody. Moreover, high glucose does not promote TGF-β accumulation in the culture media. Therefore, MAPK pathway may play an important role in high glucose-induced FN accumulation via TGF-β-independent mechanism. It is considered that high glucose acts as osmotic stimulation in the regulation of FN production in these cells. Taken together, these findings suggest that the activation of MAPK signaling pathways in rat PMCs by high glucose may contribute to the pathogenesis of peritoneal fibrosis in CAPD therapy
