CNPY2介導急性腎損傷至慢性腎病連續體的上皮間質轉化

Abstract

急性腎臟損傷(Acute Kidney Injury, AKI)在過去一被認為是一種自限性、可逆的疾病，但隨著醫療科技的進步，急性腎損傷的死亡率並未有顯著的改善，且長期追蹤這些患者在未來罹患慢性腎臟病（chronic kidney disease, CKD）的風險增加了8.8倍、進展到末期腎臟疾病（end stage renal disease, ESRD）的風險也增加了3.3倍，不僅增加患者的死亡率，也造成醫療照護成本的增加。因此，有越來越多的研究針對急性腎損傷進展到慢性腎臟病（AKI-to-CKD Transition）過程中的調控機制進行探討，企圖找出可能的治療方式。本實驗室先前的研究發現miR-30a-3p能抑制近端腎小管細胞中FN1、COL4A1等細胞外基質相關基因的表達，因此我們認為miR-30a-3p在上皮間質轉化及細胞外基質中可能扮演非常重要的角色。進一步以TargetScan分析發現CNPY2的3’UTR為其標的。CNPY2為內質網壓力PERK-CHOP pathway的啟動子，可促進癌細胞上皮間質轉化的發生並增加侵襲能力。因此，本次研究將探討CNPY2在急性腎損傷到慢性腎臟病的過程中是否扮演調控上皮間質轉化的角色。首先，我們建立小鼠單側腎臟缺血再灌流的動物模型模擬AKI-to-CKD的發展，並證實miR-30a-3p的表現下調且近端腎小管的CNPY2表現在手術後有顯著上升的情況；在細胞實驗中以TGF-β1誘發近端腎小管上皮間質轉化，發現miR-30a-3p及CNPY2的表現也與動物實驗的結果一致。除此之外，qPCR的結果中也證實miR-30a-3p確實能調控CNPY2的表現。在進一步的細胞實驗中，我們發現CNPY2會增加細胞膜中TGF-β第一型受體的表現、促進AKT的磷酸化、並增加上皮間質轉化相關蛋白的表現。為了進一步確認CNPY2是否為纖維化發展中所必須的蛋白，我們使用CNPY2 RNAi（RNA interference）抑制CNPY2的表現後讓細胞暴露於TGF-β1。然而，結果發現CNPY2表現被抑制之後並不能消除由TGF-β1誘發的上皮間質轉化。儘管如此，TGF-β1也無法顯著地讓CNPY2缺乏的近端腎小管細胞表現上皮間質轉化相關的蛋白。總而言之，在病人發展慢性腎臟疾病的過程當中miR-30a-3p的表現下降使得CNPY2無法被抑制，促使snail的增加並導致纖聯蛋白及膠原蛋白表現上升。

During the past few years, an increasing number of studies aim to investigate the regulatory mechanism during AKI-to-CKD transition. Recently, our lab investigated that miR-30a-3p, which was downregulated in fibrotic renal disease, post-transcriptionally regulated the fibrosis-associated genes in HK2 cells. Further analysis by using TargetScan revealed that miR-30a-3p might regulate CNPY2 via interacting with the mRNA 3’- untranslated region (3’UTR). CNPY2 is an ER luminal protein and a key initiator of the PERK pathway during ER stress response. In addition, researchers have demonstrated that CNPY2 enhances the progression of epithelial-mesenchymal transition (EMT) resulting in a higher invasion ability in cancer cells. Therefore, we hypothesized that CNPY2 might participate in the EMT process during the AKI-to-CKD transition. We revealed that CNPY2 was significantly increased in the kidneys of CKD patients and the fibrotic kidneys in the unilateral ischemia-reperfusion injury (UIRI) mice model and unilateral ureteral obstruction (UUO) mice model. Moreover, CNPY2 had a negative correlation with miR-30a-3p, suggesting the potential role of CNPY2 in renal fibrogenesis. According to the immunohistochemistry (IHC) staining showing that upregulated-CNPY2 was located in the proximal tubular epithelial cells, we then used the HK2 cells for the further mechanistic in vitro study. Consistent with in vivo study, CNPY2 was increased in transforming growth factor β1 (TGF-β1) treated HK2 cells. Furthermore, the real-time polymerase chain reaction (qPCR) and the western blotting analysis showed that CNPY2 overexpression activated the PERK-CHOP pathway and promoted the protein expression level of EMT-associated markers. In addition, the expression of TGFβR1 membrane protein and the phosphorylation of AKT were significantly upregulated after transfecting CNPY2 plasmid in HK2 cells. However, in in vitro study, CNPY2 knockdown via transfecting siRNA could not eliminate TGF-β1-mediated EMT and ECM progression. Despite this condition, TGF-β1 was unable to upregulate the EMT-associated proteins in CNPY2-knockdown HK2 cells as well. In conclusion, our investigations revealed a novel molecule during AKI-to-CKD transition. Further studies are required to confirm the underlying mechanisms involved in the CNPY2-mediated renal fibrosis in order to find out the potential treatment strategy.