自體顯性多囊腎疾病的小鼠模式與治療標的之研究

Abstract

自體顯性多囊腎疾病(autosomal dominant polycystic kidney disease; ADPKD)是最常見且具威脅生命的遺傳疾病之一，大多數病人主要是PKD1 的基因發生突變。運用傳統式或條件式的基因剔除法，將小鼠直系同源基因Pkd1剔除，進而重現與人類疾病表現型特徵相似的模式鼠一直尚未成功地建立。我們先前建立一個新穎的Pkd1 hypomorphic allele，其Pkd1基因之表達顯著地被降低而非完全阻斷。這些Pkd1同型合子(homozygote)突變的老鼠會迅速發展出多囊腎疾病，此支持自體顯性多囊腎是haploinsufficiency之假說。我們進一步想探討Pkd1基因之haploinsufficiency效應，透過co-cistron表達對Pkd1轉錄專一性的兩種微型核糖核酸，以建構Pkd1基因表現減量的轉殖鼠。此兩種轉基因株系的老鼠其Pkd1基因表現約減量60-70%，並且與人類自體顯性多囊腎疾病的病程相類似。這些結果進一步支持haploinsufficiency的假說，並且推測多囊腎疾病的發生與進展是與Pkd1基因表現量息息相關。因此，此兩種基因轉殖鼠可以作為研究自體顯性多囊腎疾病的理想模式鼠。 嗜中性白血球膠原蛋白質酶相關疏水性蛋白質（Neutrophil gelatinase-associated lipocalin; Ngal）是急性和慢性腎損傷(其中也包括多囊腎疾病）的生物標誌物。然而，Ngal基因對多囊腎疾病進展之影響尚未被探討。為了闡明Ngal在PKD中的作用，我們利用已建立的多囊腎疾病小鼠模式（Pkd1L3 / L3小鼠）產生了具有不同表達程度的Ngal的三種小鼠：Pkd1L3 / L3（具有內源性Ngal基因），Pkd1L3 / L3; NgalTg / Tg（具有內源性和過表達外源性腎臟專一性的Ngal基因）和Pkd1L3 / L3; Ngal - / - 小鼠（具有Ngal基因缺失）。內源性Ngal基因的剔除對Pkd1L3 / L3小鼠的表現型，囊泡進展或存活率沒有影響。然而，與具有內源性Ngal基因或Ngal基因剔除的小鼠相比，Pkd1L3 / L3; NgalTg / Tg小鼠具有顯著性更長的壽命，更小的（並非數目較少的）腎囊泡和更輕微的間質纖維化。西方墨點分析法顯示Pkd1L3 / L3; NgalTg / Tg小鼠與其他兩組PKD小鼠相比，其Ngal表現和活化的細胞凋亡蛋白質-3 (active caspase 3)有顯著性地增加，並且其α-平滑肌肌動蛋白(α-smooth muscle actin)、缺氧誘導因子-1α (hypoxia-inducible factor 1-α)、凋亡蛋白質酶原-3 (pro-caspase 3)、增殖細胞核抗原 (proliferating cell nuclear antigen)、Akt、哺乳動物雷帕霉素靶蛋白質 (mammalian target of rapamycin)和S6激酶有顯著性地減少。總之，過度表達外源性腎臟專一性的Ngal可以減少囊泡進展並延長PKD小鼠的壽命，其分子病理機轉是透過減少腎臟囊泡之生長，減緩腎間質性之纖維化，減少囊泡上皮細胞之增殖並且增加囊泡上皮細胞之細胞凋亡有關。

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening inherited diseases, and the PKD1 gene is responsible for most cases of this disease. Establishing a mouse model to recapitulate the phenotypic characteristics of ADPKD, which have used conventional or conditional knockout of the mouse orthologue Pkd1, have been unsuccessful. We previously established a novel Pkd1 hypomorphic allele, in which Pkd1 expression was significantly reduced but not totally blocked. These Pkd1 homozygous mutant mice rapidly developed renal cystic disease, supporting the hypothesis that ‘haploinsufficiency’ explains development of the ADPKD phenotype. We further explored the Pkd1 haploinsufficiency effect by generating Pkd1 knockdown transgenic mice with co-cistronic expressing of two miRNA hairpins specific to Pkd1 transcript. Two transgenic lines which had about 60 to 70% reduction of Pkd1 expression developed renal cystic disease at a rate similar to that of human ADPKD. These results further support the haploinsufficiency hypothesis, and suggest that the onset and progression of the renal cystic diseases are closely associated with the level of Pkd1 expression. These two mutant lines of mice could be ideal models for the study of ADPKD.

Neutrophil gelatinase-associated lipocalin (Ngal) is a biomarker for acute and chronic renal injuries, including polycystic kidney disease (PKD); however, the effect of Ngal on PKD progression remains unexplored. To clarify the role of Ngal in PKD, we generated three kinds of mice with different expression levels of Ngal based on an established PKD model (Pkd1L3/L3 mice): Pkd1L3/L3 (with endogenous Ngal), Pkd1L3/L3; NgalTg/Tg (with endogenous and overexpression of exogenous kidney-specific Ngal) and Pkd1L3/L3; Ngal-/- mice (with Ngal deficiency). Knockout of endogenous Ngal had no effect on phenotypes, cystic progression or survival of Pkd1L3/L3 mice. However, the Pkd1L3/L3; NgalTg/Tg mice had a significantly longer lifespan, smaller (but not fewer) renal cysts and less interstitial fibrosis than the mice with endogenous Ngal or without Ngal. Western-blot analyses showed significant increases in Ngal and active caspase-3, and decreases in α-smooth muscle actin, hypoxia-inducible factor 1-α, pro-caspase 3, proliferating cell nuclear antigen, Akt, mammalian target of rapamycin, and S6 Kinase in the Pkd1L3/L3; NgalTg/Tg mice as compared with the other two groups of PKD mice. In conclusion, overexpression of exogenous kidney-specific Ngal could reduce cystic progression and prolong the lifespan in PKD mice, and the molecular pathomechanisms were associated with reductions in cystic growth, interstitial fibrosis and proliferation, and augmentation of apoptosis.