以細胞及動物模式探討鄰苯二甲酸二(2-乙基己基)酯致腎臟纖維化之機制

Abstract

鄰苯二甲酸二(2-乙基己基)酯(di(2-ethylhexyl) phthalate, DEHP)為常用於添加在聚氯乙烯塑膠製品的塑化劑，隨塑膠製品的大量製造及使用，全球每年DEHP平均使用量已超過200萬噸，DEHP暴露於環境及生物體的頻率及劑量也隨之增加，其對環境與生物體的潛在危害性已不容忽視。目前文獻已指出DEHP對生殖系統、肝臟與腎臟具毒性，甚至國際癌症研究中心將其歸類為疑似致癌物。儘管過去研究已顯示DEHP會造成腎小管損傷與加劇腎炎病徵等，且具時間和劑量效應(time- and dose-dependent)，但對於DEHP造成腎毒性之詳細分子機制仍未釐清。末期腎臟病在台灣的高盛行率及高發生率，已造成龐大的醫療及社會成本，其中腎小管及間質纖維化是末期腎臟病的共同病理特徵，故DEHP是否會誘導或加劇腎纖維化的發生，是值得被關注的。腎小管的上皮-間質轉換過程是腎纖維化的重要路徑之一，為此本研究以腎小管上皮細胞與疾病動物模式，探討DEHP是否經由上皮-間質轉換過程，導致腎纖維化的發生，及其機制為何。實驗發現使用25 μM DEHP暴露72小時，會導致大鼠腎近曲小管上皮細胞株的外觀形態由緊密接合鵝卵石狀轉為紡錘纖維狀，細胞存活率也略降至85%。但由流式細胞儀觀察DEHP對細胞週期及細胞凋亡的影響，指出DEHP可顯著誘導細胞停滯於G2/M週期，並引起少量(0.74%)的細胞凋亡，暗示25 μM DEHP主要透過改變細胞週期以至細胞存活率下降。另一方面，於DEHP暴露下，上皮細胞指標蛋白E-cadherin表現顯著下降，間質細胞指標蛋白(CTGF、α-SMA和Vimentin)與纖維化指標分子Fibronectin表現量顯著增加。同時，25 μM DEHP會增加AKT及Smad2/3之磷酸化，並抑制PPAR-α和PPAR-γ表現量。進一步利用AKT抑制劑-MK2206探究詳細機轉，發現合併使用MK2206與DEHP，上皮-間質轉換的指標蛋白(E-cadherin及Vimentin)與纖維化指標分子(Fibronectin)，有顯著回復情形；且AKT下游蛋白NF-ĸB和GSK3磷酸化蛋白表現量也隨之改變。此外，研究也給予PPAR-γ促進劑- pioglitazone (PIO)，結果顯示合併暴露PIO與DEHP時，PPAR-γ、上皮-間質轉換指標蛋白和纖維化指標分子的表現量無顯著變化。總結上述，DEHP可能透過AKT/NF-ĸB及AKT/GSK3訊息傳遞路徑誘發腎臟上皮細胞的上皮-間質轉換與纖維化發生。本研究僅確認DEHP可經AKT訊息傳遞路徑引發上皮-間質轉換及纖維化的發生，但未釐清PPAR-γ與上皮-間質轉換過程之詳細分子機轉，以及AKT與PPAR-γ是否相互調控，此部分仍需更多實驗探究討論。動物實驗方面，以單次腹腔注射葉酸(200 mg/kg.bw)誘導腎纖維化的疾病動物模式下，探討C57BL/6公鼠持續暴露DEHP (50 mg/kg.bw/day)六週是否會引起更強烈之腎纖維化。結果指出，與單獨處理葉酸組別比較，合併處理葉酸及DEHP組別之存活率達顯著下降，而腎臟組織切片染色實驗也顯示腎小管損傷及腎纖維化(以膠原蛋白沉澱程度評估)有顯著增加的趨勢，換而言之，結果持續暴露DEHP可能會加重腎功能損傷。但在相對腎臟重與腎損傷指標(BUN、Creatinine及Cystatin C)之實驗，合併處理葉酸及DEHP組別與單獨處理葉酸組別比較下，未達統計差異。總結本篇動物研究，實驗發現持續暴露DEHP可能會影響小鼠的腎功能，進而加劇腎纖維化之情形，但仍需更多實驗與證據直接證實DEHP與腎纖維化之關係。

Di(2-ethylhexyl)phthalate (DEHP) is the major phthalate plasticizer for polyvinyl chloride. More than two million tons of DEHP were used each year worldwide. At present, DEHP has widely spread in our environment. Its potential environmental and health hazards cannot be ignored due to its low degradability. Recent studies suggest that DEHP has potential adverse effects on the liver, kidney, and reproductive organs, and causes carcinogenicity and developmental toxicity. Several studies in mice have reported dose- and time-dependent kidney lesions (ex: renal tubule injury) following exposure to DEHP. It may exacerbate chronic progressive nephropathy. The incidence of end-stage renal disease (ESRD) in Taiwan is highest in the world. In 2012, the cost of dialysis in ESRD patients was around one billion U.S. dollars, which accounted for 5.89% of the total medical expenditure of national health insurance. Those researches showed that ERSD is an important health issue in Taiwan. Tubulointerstitial fibrosis (TIF) is one of the major causes of ESRD, and tubular epithelial-to-mesenchymal transition (EMT) is recognized to play pivotal roles in the process of renal fibrosis. However, the mechanisms of nephrotoxicity induced by DEHP remain unclear.In this study, we investigated whether DEHP could induce renal fibrosis via EMT process in vitro and in vivo. After treatment with DEHP for 72h, we found that cell morphology was changed from flattened epithelial cell type to spindle-like shape under 25 μM DEHP treatment in rat renal proximal tubular cells (NRK-52E cells). Cell viability was decreased to 85.86 %. In cell cycle study, DEHP increased only 0.74 % cell apoptosis compared to control group but significantly enhanced cells which stayed in G2/M phase. These results indicated that 25 μM DEHP caused little cytotoxicity in NRK-52E cells, but induced G2/M cell cycle arrest. On the other hand, E-cadherin, a maker of epithelial cells was decreased by DEHP treatment. Vimentin、α-SMA and CTGF which are the markers of the mesenchymal phenotype were significantly increased. Furthermore, DEHP induced the protein expression of Fibronectin, a fibrosis marker. Moreover, phosphorylation of Akt and Smad 2/3 was significantly increased by DEHP. PPAR-α and PPAR-γ were decreased by DEHP treatment. Notably, treatment with MK2206 (an Akt inhibitor) significantly inhibited DEHP-induced phosphorylation of Akt and EMT. Further investigation revealed that MK2206 suppressed the expression of Akt downstream proteins (NF-ĸB and GSK3), but treatment with pioglitazone (a PPAR-γ agonist) did not reverse the expression of EMT biomarkers. Taken together, our results indicate that DEHP induce the EMT process through AKT signaling pathway and may lead to renal fibrosis in vitro. In this study we used male C57BL/6 mice to investigate the role of DEHP (50 mg/kg.bw/day) in renal fibrosis. After 4 weeks of DEHP exposure, renal fibrosis was induced by folic acid injection. The survival rate of DEHP exposure group was significantly decreased compared to vehicle group after renal fibrosis induction. The similar results were found in histological examination (H E stain and Masson's trichrome stain). Those results showed that the mice have worse renal function after DEHP treatment. The relative kidney weight and the values of blood urine nitrogen、creatinine and Cystatin C significantly increased in DEHP exposure group compared to vehicle group. However, there no significant differences between DEHP exposure group and vehicle group in kidney weight and biochemistry parameters after mice treated with folic acid. It is the first time that we found DEHP induced EMT and the expression of renal fibrosis marker through AKT pathway. We also found DEHP could aggravate folic acid-induced renal fibrosis in mice. However, we need more experiments to investigate the mechanisms of DEHP in renal fibrosis.