Ngal蛋白對自體顯性遺傳多囊腎疾病細胞模式之治療影響與機制

Abstract

多囊腎疾病(Polycystic kidney disease, PKD)為最常見的遺傳疾病之一，盛行率高達1:400~1:1000，患者主要病徵為其腎元中各個部位發展出許多充滿液體的囊泡(cysts)構造。自體顯性多囊腎疾病(Autosomal dominant polycystic kidney disease; 簡稱ADPKD)為多囊腎疾病中最常見的類型，由PKD1 (佔其中85%)或PKD2 (佔其中15%)基因發生突變所導致。PKD1與PKD2分別表現polycystin-1 (PC1)與 polycystin-2 (PC2)蛋白，而PC1或PC2的功能缺損造成腎臟上皮細胞之不正常增殖，並進而導致囊泡形成。隨著ADPKD的進程，囊泡體積增加並破壞正常腎臟組織的結構，進而造成腎功能損傷，最終導致患者之腎臟衰竭。我們先前的研究建立了ADPKD之疾病模式鼠(Pkd1L3/L3小鼠)，並利用基因轉殖技術使病鼠在腎臟中大量表現外源性neutrophil nelatinase-associated lipocalin (Ngal)，結果顯示能夠減緩囊泡的進程並延長病鼠的壽命。Ngal是一22-kD之分泌性蛋白，尿液中之Ngal含量常用作檢測患者是否具有腎臟疾病的指標，而文獻中指出Ngal與其受器Ngal receptor (Ngal-R)之交互作用與細胞內鐵離子運輸有關，並能進一步誘導細胞凋亡(apoptosis)。然而，Ngal在ADPKD中之作用機制仍未知，如用動物模式(in vivo)進行研究有許多困難，如細胞間複雜之交互作用以及研究費時等，因此在本研究中建立3D細胞平台，並直接利用ADPKD疾病細胞模式(Pkd1L3/L3)進行Ngal蛋白之作用機制研究。細胞存活實驗(MTT)與細胞增殖實驗(BrdU incorporation)之結果顯示小鼠Ngal重組蛋白(mNgal)顯著降低ADPKD細胞模式之細胞活性與增殖，並顯著地抑制囊泡生長。根據流式細胞儀分析(flow cytometry)與西方墨點法(western blot)結果顯示，小鼠Ngal重組蛋白顯著抑制ADPKD細胞中ERK與AKT訊息傳遞路徑並誘導細胞凋亡與細胞自噬(autophagy)。再者，於ADPKD細胞中大量表現分泌性小鼠Ngal蛋白顯著抑制細胞增殖與囊泡生長，但大量表現非分泌性小鼠Ngal蛋白則不具有相同之抑制效果；這些結果表示Ngal需要從ADPKD細胞中被分泌出，才能抑制細胞增殖。未來我們將利用CRISPR技術將疾病細胞之Ngal-R基因剃除，進一步探討Ngal與Ngal-R在ADPKD中之作用機制。希望有助於了解Ngal在ADPKD細胞中之治療效果與機制。

Polycystic kidney disease (PKD), one of the most common inherited diseases with a prevalence of 1:400 to 1:1000, is characterized by the development of fluid-filled cysts along multiple segments of the nephron. Autosomal dominant polycystic kidney disease (ADPKD), the most common form of PKD, is the result of mutations in either PKD1 (85%) or PKD2 (15%) genes that encode polycystin-1 (PC1) and polycystin-2 (PC2) respectively. PC1 or PC2 defect is associated with abnormal increase in proliferation of renal epithelial cells and further causes cyst formation. As ADPKD progresses, cysts enlarge and disrupt the normal kidney construction, eventually leading to kidney failure. Our previous study has showed that overexpression of exogenous kidney-specific neutrophil gelatinase-associated lipocalin (Ngal), a 22-kD secreted protein and biomarker for kidney injuries, reduced cysts progression and prolonged the lifespan of ADPKD mice (Pkd1L3/L3, 2L3 for short). It has been demonstrated that Ngal and Ngal receptor (Ngal-R) coupling could sequester intracellular iron and further induce apoptosis. However, the mechanism of effects of Ngal in ADPKD is still unclear and having multiple difficulties studying in vivo, including complex cellular interaction systems and time consuming. In this study, results of MTT assay and BrdU incorporation assay showed that recombinant mouse Ngal (mNgal) protein significantly decreased cell viability and proliferation of 2L3 cells. Results of flow cytometry analysis and western blot showed that mNgal inhibited activation of ERK and AKT pathways and induced apoptosis and autophagy in 2L3 cells. In addition, 3D cell culture platform was established for testing the cyst progression of 2L3 renal epithelial cells, which showed that mNgal significantly inhibited cyst enlargement of 2L3 cells. Overexpression of secreted mNgal (pN+LS) rather than non-secreted mNgal (pN-LS) inhibited cell proliferation and cyst enlargement in 2L3 cells through inhibiting ERK and AKT pathways and inducing apoptosis and autophagy. These results reveal that secretion of mNgal from 2L3 cells is required to inhibit cell proliferation and cyst enlargement. To research the relationship between apoptosis and coupling of Ngal and Ngal-R in ADPKD cells, knockout of Ngal-R by CRISPR in 2L3 cells is being performed. We hope to identify the therapeutic effect and mechanism of Ngal protein on ADPKD cells.