Kim Kyung-Hoon, Ryu Hye-Myung, Oh Se-Hyun, Oh Eun-Joo, Ahn Ji-Sun, Lee Jong-Hak, Choi Ji-Young, Cho Jang-Hee, Kim Chan-Duck, Kim Yong-Lim, Park Sun-Hee
Division of Nephrology and Department of Internal Medicine, Kyungpook National University Hospital, Daegu, Korea.
Ther Apher Dial. 2014 Dec;18(6):628-36. doi: 10.1111/1744-9987.12186. Epub 2014 Sep 26.
Encapsulating peritoneal sclerosis (EPS) involves excessive peritoneal fibrosis in patients on peritoneal dialysis, eventually leading to visceral constriction and bowel obstruction. Few studies have investigated epigenetic mechanisms relating to EPS. Here we evaluated the therapeutic effects of DNA demethylation in experimental EPS. Experimental EPS was induced by intraperitoneal injection of 0.1% chlorhexidine gluconate (CG) and 15% ethanol in non-uremic male Sprague-Dawley (SD) rats. Rats were divided into three groups: group C (N=5) with saline injection only, group CG (N=7) with EPS induction for 4 weeks, and chlorhexidine gluconate and azacytidine (CGA) treated group (N=7) with EPS induction for 4 weeks and 5'-azacytidine injection for the last 2 weeks. Morphometric analysis of peritoneum and immunohistochemical staining for type 1 collagen and α-smooth muscle actin (α-SMA) were performed. Expressions of transforming growth factor-β (TGF-β), fibroblast-specific protein 1 (FSP1), and DNA methyltransferase 1 (DNMT1) were analyzed by Western blot. Methylation-specific polymerase chain reaction (PCR) for Ras GTPase activating-like protein 1 (RASAL1) was performed with measurement of RASAL1 protein expression. Parietal peritoneal thickness and the number of vessels in omental tissue were significantly decreased in group CGA compared to group CG, as were the expressions of type 1 collagen, α-SMA, TGF-β, and FSP1. DNMT1 was significantly increased in group CG, and reduced in group CGA. RASAL1 hypermethylation was associated with decreased RASAL1 protein expression in group CG, which was reversed in group CGA. DNA demethylation by 5'-azacytidine treatment improved pathologic changes of the peritoneum in experimental EPS, and was associated with reversal of increased DNMT1 expression and RASAL1 hypermethylation.
包裹性腹膜硬化（EPS）见于腹膜透析患者，表现为腹膜过度纤维化，最终导致脏器受压和肠梗阻。很少有研究探讨与EPS相关的表观遗传机制。在此，我们评估了DNA去甲基化在实验性EPS中的治疗作用。通过向非尿毒症雄性Sprague-Dawley（SD）大鼠腹腔注射0.1%葡萄糖酸氯己定（CG）和15%乙醇诱导实验性EPS。大鼠分为三组：仅注射生理盐水的C组（N = 5）、诱导EPS 4周的CG组（N = 7）、诱导EPS 4周并在最后2周注射5'-氮杂胞苷的葡萄糖酸氯己定和氮杂胞苷（CGA）治疗组（N = 7）。对腹膜进行形态计量分析，并对Ⅰ型胶原和α-平滑肌肌动蛋白（α-SMA）进行免疫组化染色。通过蛋白质印迹法分析转化生长因子-β（TGF-β）、成纤维细胞特异性蛋白1（FSP1）和DNA甲基转移酶1（DNMT1）的表达。采用甲基化特异性聚合酶链反应（PCR）检测Ras GTP酶激活样蛋白1（RASAL1），并测定RASAL1蛋白表达。与CG组相比，CGA组壁层腹膜厚度和网膜组织血管数量显著降低，Ⅰ型胶原、α-SMA、TGF-β和FSP1的表达也显著降低。DNMT1在CG组显著升高，在CGA组降低。CG组中RASAL1高甲基化与RASAL1蛋白表达降低相关，而在CGA组中这种情况得到逆转。5'-氮杂胞苷处理导致的DNA去甲基化改善了实验性EPS中腹膜的病理变化，并与DNMT1表达增加和RASAL1高甲基化逆转相关。
