TW201722460A - 嗜中性白血球膠原蛋白質酶相關疏水性蛋白質之製藥用途 - Google Patents
嗜中性白血球膠原蛋白質酶相關疏水性蛋白質之製藥用途 Download PDFInfo
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Abstract
本發明關於一種嗜中性白血球膠原蛋白質酶相關疏水性蛋白質於製造藥物之用途,該藥物係用於預防或治療多囊腎疾病。
Description
本發明關於一種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)用於製造藥物之用途,該藥物係用於預防或治療多囊腎疾病(Polycystic Kidney Disease,PKD)。
常染色體顯性多囊腎疾病(Autosomal dominant Polycystic Kidney Disease,ADPKD)為常見的遺傳疾病中之一,盛行率約莫四百分之一至千分之一,大概有85%左右的病患係因位於第十六號染色體之PKD1基因發生突變(Torres VE,et al.,Lancet,369:1287-1301,2007;Grantham JJ,et al.,Nat Rev Nephrol,7:556-566,2011)。PKD1係表現出聚胱胺酸-1(polycystin-1,PC1),實驗小鼠顯示胚胎時期PC1表現量下降與早期囊泡形成及疾病嚴重度有關(Piontek K,et al.,Nat Med,13:1490-1495,2007;Hopp K,et al.,J Clin Invest,122:4257-4273,2012;Fedeles SV,et al.,Trends Mol Med,20:251-260,2014;Lu W,et al.,Nat Genet,17:179-181,1997;Magenheimer BS,et al.,J Am Soc Nephrol,17:3424-3437,2006)。
患者一般於四十歲左右發病,平均在五十歲左右即到達腎功能衰竭的程度,因而需依賴血液透析或腹膜透析等透析技術或腎臟移植,
才能夠延續生命。ADPKD屬於自體顯性遺傳疾病,換言之,父母其中一人患病,小孩就有一半機會罹患此疾。全世界目前約有一千兩百萬個ADPKD病患,美國ADPKD盛行率約五百分之一,約有六十萬人罹患此病,其中約有50% ADPKD病患在五十歲就發生腎衰竭,此疾病也是造成慢性腎功能衰竭的第四大肇因。
日本大塚製藥研發出的新藥Tolvaptan,其為抗利尿激素受體拮抗劑,可用於治療心臟衰竭引起之低血鈉症,大塚製藥意外發現其對ADPKD的小鼠和大鼠之動物模式具有療效,進而舉行約1445個ADPKD病患參與的全球大規模人體實驗,結果顯示Tolvaptan雖可有效降低多囊腎體積及腎臟功能低落,但是卻有不可逆的肝臟毒性。
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),是個22kD之分泌性蛋白質,其自胚胎時期開始表現於腎單元,且涉及腎臟發育(Yang J,et al.,Mol Cell,10:1045-1056,2002)及對抗細菌感染的先天免疫反應(Flo TH,et al.,Nature,432:917-921,2004;Berger T,et al.,Proc Natl Acad Sci,103:1834-1839,2006)。目前研究顯示,比起傳統的肌酸酐和尿素氮(blood urea nitrogen,BUN)等生物標記(bio-markers),NGAL更為敏銳且更能早期偵測出急、慢性腎臟損傷(Mishra J,et al.,J Am Soc Nephrol, 14:2534-2543,2003;Mishra J,et al.,Lancet 365:1231-1238,2005;Urbschat A,et al.,Eur J Clin Invest, 44:652-659,2014;Di Grande A,et al.,Eur Rev Med Pharmacol Sci, 13:197-200,2009;Devarajan P,Biomark Med, 8:217-219,2014;Nickolas TL,et al.,Kidney Int, 82:718-722,2012;Shen SJ,et al.,Nephrology(Carlton), 19:
129-135,2014),亦能測出多囊腎疾病是否惡化(Parikh CR,et al.,Kidney Int, 81:784-790,2012;Meijer E,et al.,Am J Kidney Dis, 56:883-895,2010)。
NGAL受體(NGAL-R,Slc22a17)表現在集尿管及遠曲小管的頂膜且涉及內噬鐵轉運(endocytic iron delivery;Langelueddecke C,et al.,J Biol Chem, 287:159-169,2012),其與NGAL一起促成胞內鐵耗盡、刺激細胞凋亡及降低細胞增殖(Devireddy LR,et al.,Cell, 123:1293-1305,2005;Schmidt-Ott KM,et al.,J Am Soc Nephrol, 18:407-413,2007;Devarajan P.,Cancer Ther, 5:463-470,2007)。
已公開之專利文獻中,美國專利申請案公開號2014/0079769 A1揭示偵測Ngal基因表現以預測慢性腎臟病,並教示藉由抑制Ngal基因表現可預防或治療慢性腎臟病(chronic kidney disease,CKD);中國大陸專利申請案公開號101163971 A揭示經由測量患者體液樣本中的NGAL含量,藉此判斷並監測患者的腎臟情況;美國專利申請案公開號2011/0091912 A1揭示經由測量NGAL高分子量型含量,可區別CKD或急性腎損傷或腎臟病(acute kidney injury or disease,AKI或AKD)。
中國大陸專利申請案公開號101010001 A揭示通過靜脈注射、皮下給藥或腹膜內,將NGAL投予局部缺血、局部缺血再灌注、或毒素引起的損傷和急性或慢性腎疾病患者,顯示NGAL直接地靶向腎近端腎小管細胞,其可增強腎上皮再形成(re-epithelialization),改善腎小管細胞增殖及腎小管細胞凋亡,降低局部缺血再灌注損傷後高的血清或血漿肌酸酐含量。
據此,雖有相關文獻揭示局部缺血、局部缺血再灌注、或毒素引起的損傷和急性或慢性腎疾病患,可使用NGAL作為該些腎病的治療劑,但未有任何文獻教示多囊腎疾病之不具副作用的治療劑或治療方法。
鑑於多囊腎疾病尚未出現有效且不具肝副作用之藥物,本發明之發明人欲找尋對多囊腎疾病不具肝毒性副作用之藥物,因而完成本發明。過程中,發明人建立多囊腎疾病小鼠模型Pkd1 L3/L3 、腎小管專一性高度表現NGAL小鼠模型Ngal Tg/Tg ,將此兩種基因型的小鼠雜交獲得Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠,換言之該小鼠天生患有多囊腎疾病,且在胚胎階段開始至成鼠的腎小管,都會高度表達NGAL。利用該小鼠模型評估高度表現外源性且具腎專一性之NGAL對於多囊腎疾病的影響,發現高度表現外源性且具腎專一性之NGAL可以預防或治療多囊腎疾病,進而可根據此發現而開發蛋白質藥物以治療多囊腎疾病。
本發明係關於一種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)用於製造藥物之用途,該NGAL藥物係用於預防或治療多囊腎疾病(Polycystic Kidney Disease,PKD)。
本發明亦關於一種多囊腎疾病之治療或預防方法,其係將有效劑量之NGAL投遞多囊腎疾病基因突變者。本發明亦關於一種多囊腎疾病之基因治療或預防方法,其係透過基因轉殖方式,將外源性且高度表現NGAL蛋白質的基因,轉殖至多囊腎疾病基因突變者。該多囊腎疾病基因突變者係指患者具有發展成多囊腎疾病的潛力,例如多囊腎疾病尚未發病
之潛在患者或朝向多囊腎疾病發展之患者,或者已經確診患有多囊腎疾病之患者,該患者係指人類或其他動物。
本發明亦關於一種PKD/NGAL動物模型,該動物模型中表現低量的全長PC1,且高度表現外源性NGAL。本發明亦關於一種PKD/NGAL動物模型之生產方法,包含以下步驟:雜交體內表現低量全長PC1的動物與高度表現外源性NGAL的動物。
於本發明一具體實例中,該動物模型為C57BL/6J基因背景的小鼠模型,且其基因型為Pkd1 L3/L3 ;Ngal Tg/Tg ,其係透過Pkd1 L3/L3 小鼠及Ngal Tg/Tg 小鼠雜交而得。其中外源性Ngal基因係在腎小管專一性鈣黏蛋白質(kidney tubular-specific cadherin 16,Ksp-Cdh16)啟動子之控制下表現NGAL,該Ngal基因可為任何已知生物之Ngal基因,不論是否經過修飾,只要能夠表現出功能完整之NGAL即可。所述高度表現外源性NGAL係指針對腎臟NGAL/GAPDH比例而言,例如Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠腎臟之NGAL濃度為Pkd1 L3/L3 小鼠之2.6倍,為Ngal Tg/Tg 小鼠之3.8倍;所述低量PC1之表現量,係指該PC1之含量為野生型之20%以下,較佳為15%以下,更佳為10%以下。該動物模型可用於後續研究探討多囊腎疾病與NGAL及其他酵素、受體、因子、藥物等之關聯性。
除此之外,該Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠的壽命顯著性地比Pkd1 L3/L3 小鼠壽命延長為1.6至6倍,較佳為2至5倍;該Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠的腎臟/體重比例為10%-17%,更佳為12.2%-15.5%。
該NGAL可運用動物基因轉殖技術,將NGAL大量表達於牛乳或雞、鴨蛋中,或是運用植物基因轉殖技術,將NGAL大量表達於
葉、果實、種子、根、莖可食用部位,也可以將表達NGAL之載體轉型入大腸桿菌或轉染細胞株CHO,大量生產與純化NGAL藥物。
該NGAL的投遞方式,可為口服、靜脈注射、腹腔注射、皮下注射等,多樣性給予NGAL。以靜脈注射為例,NGAL或其衍生之蛋白質藥物可進入血液循環系統達到腎臟部位之吸收,降低因腸胃道酵素分解(enzymatic degradation)或肝臟分解(first-pass effect)而造成的藥效降低。此外,本發明提供之NGAL或其蛋白質藥物衍生物,相較於傳統小分子藥物如Tolvaptan,更可提供高度專一性、降低副作用及毒性、高生物相容性等優勢。
上述多囊腎疾病包含常染色體顯性多囊腎疾病(Autosomal Dominant Polycystic Kidney Disease,ADPKD)以及常染色體隱性多囊腎疾病(Autosomal Recessive Polycystic Kidney Disease,ARPKD)。本發明之具體實施例中,該多囊腎疾病為ADPKD。
對於多囊腎疾病之預防或治療,係指防止、抑制、減緩、治癒、改善該疾病。本發明之具體實例中,透過抑制囊泡擴大、減緩間質性纖維化、增加腎細胞細胞凋亡速度、減緩腎細胞增殖速度等達到治療的效果,並延長患者壽命。對於該疾病之預防,可針對具有多囊腎疾病基因突變者,於體內尚未表現內源性NGAL之胚胎時期或尚未發病時,即針對該些可能發展成多囊腎疾病之患者投遞外源性NGAL,以期達到預防之效果。
對於具有多囊腎疾病患者投遞外源性NGAL,可透過基因轉殖表現高量之NGAL或直接使用NGAL藥物投遞至患者體內,該NGAL可增加活化細胞凋亡蛋白質-3(active-caspase-3)、減少α-平滑肌肌動
蛋白質(α-SMA)、降低缺氧誘導因子-1α(hypoxia-inducible factor 1-α,HIF-1α)、減少凋亡蛋白質酶原-3(pro-caspase 3)、降低增殖細胞核抗原(proliferating cell nuclear antigen,PCNA)、減少Akt、降低哺乳動物雷帕霉素靶蛋白質(mammalian target of rapamycin,mTOR)、減少S6激酶(S6K)等,後敘三者即降低Akt-mTOR-S6K的訊息傳導途徑,以預防或治療多囊腎疾病。
由本發明之具體實例可知,該NGAL為劑量依賴性(dose-dependent),換言之,患者體內的總NGAL量需高於原本內源性NGAL總量才可能達到治療或預防多囊腎疾病之效果。
圖1A至圖1D顯示產生Ngal缺失小鼠(KO)與Cdh16-mNgal基因轉殖小鼠之示意圖;其中圖1A為針對Ngal基因座進行同源重組之示意圖;圖1B顯示Ngal轉殖基因之建構示意圖,其中經由鼠類Cdh16腎專一性啟動子控制過度表現;圖1C及1D顯示以PCR分析確認基因型Ngal -/-及Ngal Tg/Tg。
圖2A至2D顯示以西方墨點分析Pkd1 L3/L3 小鼠與野生型小鼠腎臟中NGAL的時序表現量;圖2A為NGAL表現量之西方墨點分析圖,其中顯示Pkd1 L3/L3 小鼠與野生型小鼠之腎臟、尿及血清中NGAL的表現量;圖2B、2C及2D顯示西方墨點分析定量結果,其中β-肌動蛋白作為內控制組,數據皆為平均±標準誤差;n=6;**為p<0.01;***為p<0.001。
圖3顯示以免疫組織化學分析Pkd1 L3/L3 小鼠與野生型小
鼠腎臟中NGAL的時序表現,其中野生型及同型合子之不同時期腎切片,以NGAL抗體染色;(A-C)為第1天(1D)之腎切片,褐染位置顯示野生型(A)之NGAL位於髓質(medulla)而非腎小球(glomerulus)及皮質小管(cortical tubules),Pkd1 L3/L3 小鼠之NGAL位於部分的皮質(B)及髓質(C);(D-F)為第14天(14D)之腎切片,NGAL表現量於野生型(D)中較第1天降低,但於Pkd1 L3/L3 小鼠皮質(E)及髓質(F)較第1天增加;(G-I)為第28天(28D)之腎切片,囊泡較第14天(14D)Pkd1 L3/L3 小鼠(E及F)增加。在囊泡增大的期間,立方上皮轉型成鱗狀圖樣,同時亦表現NGAL(E,F,H,I),所有代表圖皆有兩次獨立試驗且每一基因型皆至少有3隻小鼠,(A-I)比例尺為200μm,(B,C,E,F,H,I)比例尺為50μm。
圖4顯示ADPKD與ARPKD病患腎組織之NGAL表現,(A、B)為人類正常腎組織,(C、D)為ADPKD腎組織,(E、F)為ARPKD腎組織,三角箭頭處指向褐染位置,代表ADPKD與ARPKD腎切片中NGAL位置,長箭頭指向ADPKD間質區(interstitium area)單核細胞中表現之內源性NGAL。(A)比例尺為200μm,(B)比例尺為50μm,(C、E)比例尺為500μm(D、F)比例尺為100μm。
圖5A至圖5C顯示Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠NGAL在腎臟的表現量,高於Pkd1 L3/L3 小鼠與Pkd1 L3/L3 ;Ngal -/-小鼠,其中圖5A為不同基因型之出生後第21天(21D)小鼠體內,NGAL、NGAL-R及3-磷酸甘油醛脫氫酶(Glyceraldehyde 3-phosphate dehydrogenase,GAPDH,內控制組)的西方墨點法代表圖,圖5B及圖5C分別為NGAL及
NGAL-R西方墨點代表圖的定量結果,其中誤差條表示三種小鼠個別的平均±標準誤差(SEM,***p<0.001)。
圖6A至圖6B顯示高度表現外源性腎專一性NGAL延長Pkd1 L3/L3 小鼠的存活時間,其中圖6A為Kaplan-Meier分析Pkd1 L3/L3 小鼠(空心三角)、Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠(實心圓)及Pkd1 L3/L3 ;Ngal -/-小鼠(空心方形)的存活率,對數等級檢定顯示Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠與Pkd1 L3/L3 小鼠之間,以及Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠與Pkd1 L3/L3 ;Ngal -/-小鼠之間,顯著具有差異(p<0.001);圖6B顯示中位數存活天數,其中Pkd1 L3/L3 小鼠為25天(n=31),Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠為46天(n=45),Pkd1 L3/L3 ;Ngal -/-小鼠為26天(n=32),所有結果皆以中位數及25%-75%範圍呈現,***p<0.001。
圖7A至圖7D顯示高度表現外源性腎專一性NGAL減小Pkd1 L3/L3 小鼠的腎囊泡,其中圖7A為各基因型的腎代表圖;圖7B顯示Dunn’s多重比較檢定第21天小鼠腎重量對身體重量比例的中位數百分比,Pkd1 L3/L3 小鼠為23.1%(n=10),Pkd1 L3/L3 ;NgalTg/Tg小鼠為14.1%(n=9),Pkd1 L3/L3 ;Ngal-/-小鼠為24.4%(n=9);圖7C顯示小鼠囊泡體積,Pkd1 L3/L3 小鼠為430.1μm2,Pkd1 L3/L3 ;NgalTg/Tg小鼠為124.0μm2,Pkd1 L3/L3 ;Ngal-/-小鼠為339.8μm2;圖7D顯示小鼠囊泡數目,所有結果皆以中位數及25%-75%範圍呈現,***p<0.001。
圖8A至圖8B顯示高度表現外源性腎專一性NGAL降低Pkd1 L3/L3 小鼠的間質性纖維化,其中圖8A顯示蘇木素及伊紅(H&E)染色(左欄)及麻森氏三色(Masson’s trichrome)染色(右欄),比例尺為
200μm;圖8B顯示由麻森氏三色染色腎臟評估的腎纖維化分數,計算方式如實施方式所述,所有結果皆以中位數及25%-75%範圍呈現;圖8C顯示西方墨點代表圖(上排)及α-SMA定量結果(下排左)與HIF-1α定量結果(下排右),柱狀圖顯示三樣本的平均±SEM,*p<0.05、**p<0.01、***p<0.001。
圖9A至圖9C顯示高度表現外源性腎專一性NGAL減少Pkd1 L3/L3 小鼠的增殖細胞核抗原(proliferating cell nuclear antigen,PCNA)及凋亡蛋白質酶原-3(pro-caspase 3)在腎臟的表現量,其中圖9A為第21天不同基因型小鼠,其PCNA、凋亡蛋白質酶-3、3-磷酸甘油醛脫氫酶(Glyceraldehyde 3-phosphate dehydrogenase,GAPDH,內控制組)的西方墨點分析代表圖;圖9B為PCNA/GAPDH之西方墨點分析定量結果;圖9C為凋亡蛋白質酶原-3/GAPDH之西方墨點分析定量結果,柱狀圖顯示三樣本的平均±SEM,**p<0.01、***p<0.001。
圖10A至圖10D顯示高度表現外源性腎專一性NGAL減少Pkd1 L3/L3 小鼠的磷酸化-哺乳動物雷帕霉素靶蛋白質(phospho-mammalian target of rapamycin,p-mTOR)及mTOR表現量,其中圖10A為第21天不同基因型小鼠,其磷酸化-mTOR(Ser2448)、mTOR及β-肌動蛋白質(內控制組)的西方墨點分析代表圖;圖10B、圖10C及圖10D分別為磷酸化-mTOR/β-肌動蛋白質、mTOR/β-肌動蛋白質及磷酸化-mTOR/mTOR之西方墨點分析定量結果,柱狀圖顯示三樣本的平均±SEM,*p<0.05、***p<0.001。
圖11A至圖11C顯示高度表現外源性腎專一性NGAL
減少Pkd1 L3/L3 小鼠的磷酸化-Akt、Akt、磷酸化-S6K及S6K在腎臟的表現量,其中圖11A為第21天不同基因型小鼠,其磷酸化-Akt(Ser473)、Akt、磷酸化-S6K(Thr389)、S6K及β-肌動蛋白質(內控制組)的西方墨點分析代表圖;圖11B及圖11C分別為磷酸化-Akt/β-肌動蛋白質及磷酸化-S6K/β-肌動蛋白質之西方墨點分析定量結果,柱狀圖顯示三樣本的平均±SEM,*p<0.05、**p<0.01、***p<0.001。
圖12A至圖12D係顯示Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠腎的磷酸化-EGFR及EGFR的表現量高於Pkd1 L3/L3 小鼠及Pkd1 L3/L3 ;Ngal -/- 小鼠,其中圖12A為第21天不同基因型小鼠,其磷酸化-EGFR(Tyr1068)、EGFR及GAPDH(內控制組)的西方墨點分析代表圖;圖12B、圖12C及圖12D分別為為磷酸化-EGFR/GAPDH、EGFR/GAPDH及磷酸化-EGFR/EGFR之西方墨點分析定量結果,柱狀圖顯示三樣本的平均±SEM,**p<0.01、***p<0.001。
<<實施例>>
臨床人類腎臟樣本及病人
所有腎臟組織獲自台灣國立成功大學醫院之人類生物銀行,此研究係經由國立成功大學醫學中心的試驗審查委員會認可(A-ER-101-228)。獲自台灣國立成功大學醫院病理部之三組人類腎臟樣本(兩組多囊腎疾病患者之腎臟、一組正常腎臟),其NGAL表現量經由免疫組織化學法(immunohistochemistry)測定。正常組織取自已故病人,其屍體
經確診沒有患有非秘尿生殖器疾病;兩組多囊腎疾病患者之腎臟中,其中一者患有常染色體顯性多囊腎疾病(ADPKD)及嚴重的腎小管間質性腎炎造成的慢性腎衰竭,另一者為30週妊娠期中止之早產兒,其具有常染色體隱性多囊腎疾病(ARPKD)及多囊巨腎臟(polycystic mega-kidney)。
免疫組織化學法
摘除腎臟並在4℃下以10%福馬林固定過夜,而後脫水嵌埋於石蠟,切成4μm片段以免疫染色。為了偵測NGAL,腎臟切片在去石蠟(deparaffinization)及復水(rehydration)後,以卵白素/生物素填阻套組(Avidin/Biotin Blocking Kit,Vector Laboratories,Burlingame,CA)處理,再以兔子抗NGAL抗體或山羊抗人類NGAL抗體於4℃下浸置過夜。對於其他免疫染色,使用標準免疫過氧化酶法(immunoperoxidase protocol,Vectastain ABC kit;Vector Laboratories)。切片以山羊血清填阻(blocking)後,在室溫下於一抗(primary antibodies)中浸置1小時,以PBS清洗,而後浸置於生物素化山羊抗兔子抗體,再清洗後浸置於鍵結有鏈卵白素(streptavidin)之過氧化酶,清洗後浸置於作為呈色劑之3-胺基-9-乙基咔唑(3-amino-9-ethyl-carbazole),以蘇木精複染,最後使用光學顯微鏡觀察。
動物實驗
所有小鼠皆在12小時晝夜循環節律下,飼養於台灣台南國家實驗研究院動物中心(National Laboratory Animal Center,NLAC),且所有實驗皆遵照NLAC實驗動物照護及使用委員會(Institutional Animal Carea nd Use Committee of NLAC)認可之方法進行,本發明中公、母小鼠
實驗犧牲前,兩者未具有顯著差異,且亦有多篇文獻同時使用兩種性別的動物進行實驗,此顯示在多囊腎疾病及分子分析上幾乎不具有差異,因此兩種性別的小鼠皆可用於本發明之實驗。
PKD小鼠
發明人先前的研究建立多囊腎疾病小鼠模型Pkd1 L3/L3 ,該小鼠生產的聚胱胺酸-1(polycystin-1,PC1)量低,因而逐漸發展成多囊腎疾病(Jiang ST,et al.,Am J Pathol,168:205-220,2006)。原始Pkd1 L3/+小鼠為C57BL6-129雜交背景之小鼠與C57BL/6J小鼠回交超過10代,獲得遺傳基因背景一致的原始Pkd1 L3/+小鼠後,使用該Pkd1 L3/+小鼠與C57BL/6J小鼠飼育同型合子突變系Pkd1 L3/L3 小鼠。
Ngal缺失小鼠
使用VelociGene生物技術(Valenzuela DM,et al.,Nat Biotechnol,21:652-659,2003)產生傳統Ngal基因缺失小鼠,以lacZ作為報導基因,使用PGK/Neo基因匣破壞小鼠Ngal基因。在產生Ngal -/-之前,原始C57BL6-129雜交背景之小鼠與C57BL/6J小鼠回交超過10代,確保Ngal -/-小鼠擁有一致的遺傳基因背景。傳統Ngal基因剔除技術可防止腎臟及其他組織生產NGAL,此可用於判定NGAL在多囊腎疾病中所扮演的功能。
Cdh16-mNgal基因轉殖小鼠
Cdh16-mNgal基因轉殖小鼠在腎小管專一性鈣黏蛋白質(kidney tubular-specific cadherin 16,Ksp-Cdh16)啟動子控制下(Shao X,et al.,J Am Soc Nephrol,13:1824-1836,2002)表現NGAL,該Ksp-Cdh16啟
動子可確保在多囊腎疾病小鼠胚胎早期內源性NGAL尚未表現且尚未形成有囊泡時,即高度表現外源性NGAL。Cdh16-mNgal基因轉殖小鼠之基因背景為C57BL/6J,其基因重組的方法係經NLAC實驗動物照護及使用委員會認可,其中Ngal係NCBI Gene No.16819或EMBL-EBI No.ENSMUSG00000026822。
基因型鑑定
使用聚合酶連鎖反應(Polymerase chain reaction,PCR),鑑定萃取自所有突變小鼠尾巴的DNA基因組之基因型,其中每個樣本含有20μL PCR反應混合液,β-肌動蛋白質作為內控制組(internal control),小鼠Ngal引子對序列中正向引子為SEQ ID No.1(5'-ATGGCCCTGAGTGTCATGTGTC-3'),反向引子為SEQ ID No.2(5'-GCTCCAGATGCTCCTTGGTATG-3');β-肌動蛋白質引子對序列中正向引子為SEQ ID No.3(5'-GGCATTGTTACCAACTGGGACG-3'),反向引子為SEQ ID No.4(5'-AGGAAGGCTGGAAAAGAGCC-3')。使用以下引子分析Ngal缺失之突變鼠:小鼠Ngal 5' UTR正向引子序列SEQ ID No.5(5'-TTCCTCCTCCAGCACACATCAGAC-3')、lacZ反向引序列SEQ ID No.6(5'-GAGTAACAACCCGTCGGATTCTC-3')及小鼠Ngal反向引子序列SEQ ID No.7(5'-AGGGGTTACTGTCAGAGTGGCTATC-3')。
西方墨點分析(Western-Blot Analysis)
以萃取自小鼠腎臟之總蛋白質(50μg)進行西方墨點分析,其中使用pQE蛋白質表現系統(Qiagen)表現全長的小鼠NGAL並藉由6×His-tags純化NGAL,使用該經純化的小鼠NGAL對兔子進行免
疫,以獲得NGAL抗體。其他抗體為抗Slc22A17之兔子多株抗體(GTX85032,GeneTex)、GAPDH之兔子多株抗體(631401,BioLegend)、抗α-平滑肌肌動蛋白質(α-SMA)之小鼠單株抗體(A2547,Sigma-Aldrich)、抗缺氧誘導因子1-α(hypoxia-inducible factor,HIF-1α)之兔子多株抗體(GTX 127309,GeneTex)、抗磷酸(Ser2448)-mTOR之兔子單株抗體(5536,Cell Signaling Technology)、抗mTOR之兔子單株抗體(2983,Cell Signaling Technology)、抗β-肌動蛋白質(8H10D10)之小鼠單株抗體(12262,Cell Signaling Technology)、抗磷酸(Thr389)-S6K1之兔子多株抗體(ab2571,Abcam)、抗p70 S6激酶之兔子單株抗體(2708,Cell Signaling Technology)、抗磷酸(Ser473)-Akt之兔子多株抗體(9271,Cell Signaling Technology)、抗Akt之兔子多株抗體(9272,Cell Signaling Technology)、抗凋亡蛋白質酶-3之兔子多株抗體(9662,Cell Signaling Technology)、抗增殖細胞核抗原(PCNA)之小鼠單株抗體(307902,Biolegend)、抗磷酸(Tyr1068)-上皮生長因子受體(EGFR)之兔子多株抗體(2234,Cell Signaling Technology)及抗EGFR之兔子單株抗體(GTX61503,GeneTex)。
組織學及組織形態學分析
如先前研究所述(Wang,E.et al.,J Pathol,222:238-248,2010),樣本以福馬林固定,嵌埋於石蠟,切成4μm片段,使用蘇木素及伊紅(H&E)染色或麻森氏三色(Masson’s trichrome)染色,並以光學顯微鏡(Eclipse E600 Nikon,Japan)觀察。再尋求不知小鼠基因型的研究人員,定量H&E染色所顯現的囊泡數目與大小以及染色膠原纖維(Masson’s
trichrome),其經由影像處理軟體(Image-Pro Plus v.4.5.0.29 software,Media Cybemetics,Rockville,MD,20850 USA),計算平均即時分數(mean quick scores,QSs),其中每一片切片,軟體會隨機選擇及分析五個區域(200×),在適當地白平衡後,軟體允許操作者分隔出特別感興趣的區域並擷取數位影像個別分析。每一片切片經分析後,則會顯現標記指數(label index,LI)以及平均光密度(mean optical density,MOD),標記指數表示陽性染色區域對總區域的比例,平均光密度表示基於陽性畫素數的染色濃度,QS為LI與MOD的乘積。為了計算囊泡,使用H&E染色的腎橫斷切片的代表圖,其中包含腎皮質、腎髓質及腎乳突。格線覆蓋於影像上,藉由格線交叉的百分比,可分別用以計算囊泡及非囊泡區域。
統計分析
比較Pkd1 L3/L3 、Pkd1 L3/L3 ;Ngal Tg/Tg 、Pkd1 L3/L3 ;Ngal -/-等三群組,因非常規分布,存活時間以中位數及25%-75%範圍表示,由非參數Kruskal-Wallis檢定及Dunn’s多重比較檢定來比較群組。建構存活曲線,並使用Kaplan-Meier估計法,比較以對數等級檢定獲得之不同群組的存活率。使用西方墨點影像定量結果,藉由單因子變異數分析(one-way ANOVA)比較所有群組,接著每對皆進行Tukey多重比較檢測,p值低於0.05則視為統計上具有顯著意義。
<實驗結果>
產生傳統Ngal缺失小鼠及Cdh16-mNgal基因轉殖小鼠
針對小鼠的Ngal標靶策略(圖1A)及Cdh16-mNgal基因轉殖小鼠之建構(圖1B),使用PCR分析確定該些小鼠的基因型(圖
1C)。將Cdh16-mNgal基因轉殖小鼠之同型合子Ngal Tg/Tg 小鼠與野生型C57BL/6J小鼠回交後,確認其後代皆為異型合子Ngal Tg/o 小鼠,即可驗證同型合子。此外,Ngal Tg/o 及NgalT g/Tg 小鼠其Ngal基因表現程度與Ngal的拷貝數目成正相關(圖1D)。
NGAL在Pkd1
L3/L3
;Ngal
Tg/Tg
小鼠腎臟中表現量皆增加
Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠係利用Pkd1 L3/+;Ngal Tg/Tg 小鼠互相雜交獲得。以西方墨點分析,檢視野生型(WT)小鼠及Pkd1 L3/L3 小鼠其尿液、血清及腎臟組織中內源性NGAL之表現量(圖2A至圖2D)。結果證明在Pkd1 L3/L3 小鼠其NGAL之表現量隨著囊泡逐漸增大而增加;反之,在野生型小鼠其NGAL之表現量隨著年齡增加而減少。以免疫組織化學分析分別檢視出生後第1天(1D)、第14天(14D)、第28天(28D)之野生型及Pkd1 L3/L3 小鼠其腎臟組織之NGAL表現位置與量圖譜(圖3)。第14天(14D)及第28天(28D)的Pkd1 L3/L3 小鼠中,內源性NGAL主要位於囊泡上皮細胞的頂面,且Pkd1 L3/L3 小鼠腎髓質的囊泡隨著年齡增長而增大。在常染色體顯性多囊腎病或常染色體隱性多囊腎病(autosomal recessive polycystic kidney disease,ARPKD)的病患腎臟中,可觀察到與Pkd1 L3/L3 小鼠類似的NGAL表現位置圖譜(圖4)。本發明的研究結果顯示,囊泡上皮之內源性NGAL表現量異常地增加,可能反映出腎臟異常的發育,且該些證據亦顯示NGAL與多囊腎疾病之嚴重度成正相關。其次,透過西方墨點法檢視第21天大的三組多囊腎疾病小鼠(即Pkd1 L3/L3 小鼠、Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠、Pkd1 L3/L3 ;Ngal -/-小鼠)其腎臟組織中NGAL及NGAL-R之表現(圖5A),其中Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠NGAL之表現量分別為
Pkd1 L3/L3 小鼠與Ngal Tg/Tg 小鼠的1.8倍及3.7倍(圖5B);此外,相較於其個別對應之同窩非多囊腎疾病之對照組小鼠,三組多囊腎疾病小鼠的NGAL-R,在腎臟細胞表現量顯著地提高(p<0.001,圖5A及5C)。上述結果亦顯示,在該Ksp-Cdh16啟動子的控制下,外源性NGAL係在內源性NGAL尚未表現之胚胎早期,即持續高度表現,可於胚胎時期即預防Pkd1 L3/L3 小鼠朝向多囊腎疾病發展。
高度表現Cdh16-mNgal延長Pkd1
L3/L3
;Ngal
Tg/Tg
小鼠存活時間
藉由差別表現NGAL,分析NGAL對於Pkd1 L3/L3 小鼠、Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠、Pkd1 L3/L3 ;Ngal -/-小鼠的影響(圖6A),對數等級檢定顯示Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠的壽命(中位數為46天,25%-75%範圍(IQR)為37-60天)比起Pkd1 L3/L3 小鼠的壽命(中位數為25天,IQR為23-26天)以及Pkd1 L3/L3 ;Ngal -/-小鼠的壽命(中位數為26天,IQR為24-31天)顯著地較長(p<0.001,圖6B)。此外,Pkd1 L3/L3 小鼠與Pkd1 L3/L3 ;Ngal -/-小鼠之間,壽命並無顯著差異(p=0.2988)。
高度表現Cdh16-mNgal減緩Pkd1
L3/L3
;Ngal
Tg/Tg
小鼠囊泡增大
目前已知腎臟體積總和(TKV)與尿素氮(BUN)之增加與多囊腎疾病之進展呈正相關。21天大的Pkd1 L3/L3 小鼠及Pkd1 L3/L3 ;Ngal -/-小鼠其多囊性腎臟大約發展至末期。同樣21天大的小鼠,Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠比起Pkd1 L3/L3 小鼠及Pkd1 L3/L3 ;Ngal -/-小鼠,其腎臟尺寸明顯較小(圖7A);且依腎臟重量與體重的比例(以中位數表示),Pkd1 L3/L3 ;Ngal Tg/Tg 小
鼠(中位數為14.1%,IQR為12.2%-15.5%)比起Pkd1 L3/L3 小鼠(中位數為23.1%,IQR為20.9%-27.6%)及Pkd1 L3/L3 ;Ngal -/-小鼠(中位數為24.4%,IQR為22.7%-27.7%)更顯著地(p<0.001)減少(圖7B);依腎囊泡尺寸統計,Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠比起其他兩種多囊腎疾病小鼠亦顯著較小(p<0.001)(圖7C),然而囊泡的數目卻沒有統計學上的差異(圖7D)。這些結果表示高度表現外源性、腎專一性的NGAL,可避免多囊腎疾病的病程快速進展,且顯著地減少Pkd1 L3/L3 小鼠的囊泡尺寸而非囊泡數目。由上述結果可知,Pkd1 L3/L3 小鼠中剔除Ngal基因(Pkd1 L3/L3 ;Ngal -/-小鼠),多囊腎疾病並未受影響,此可能是因為多囊腎疾病的進程係取決於PC1減少而非NGAL減少。此外,Pkd1 L3/L3 小鼠中即使具有內源性的NGAL,亦無法保護小鼠早期免於死亡。
高度表現Cdh16-mNgal減少腎臟組織間質性纖維化
圖8A顯示第21天腎臟切片的蘇木素及伊紅(H&E)染色(左欄)及麻森氏三色(Masson’s trichrome)染色(右欄),其中野生型小鼠的腎臟纖維化分數顯著低於Pkd1 L3/L3 小鼠(p=0.002)及Pkd1 L3/L3 ;Ngal -/-小鼠(p=0.015),但與Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠(p=0.417)沒有顯著差異(圖8B)。進一步檢視與小鼠腎臟纖維化及囊泡生長有關的α-SMA及HIF-1α之表現量。西方墨點分析證明野生型小鼠與Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠中α-SMA之表現量相似(圖8C上排),然而Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠中α-SMA及H-IF-1α之表現量顯著低於其他兩種多囊腎疾病小鼠(p<0.001,圖8C下排),因此α-SMA及HIF-1α之表現量與這些小鼠的纖維化分數具有高度相關。
Pkd1
L3/L3
;Ngal
Tg/Tg
小鼠中增殖細胞核抗原(PCNA)及凋亡蛋白質酶-3在腎臟的表現量
在早期形成囊泡的多囊性腎臟中,增殖及凋亡兩者皆提升。西方墨點分析(圖9A)顯示,相較於其他兩種多囊腎疾病小鼠,Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠的增殖細胞核抗原(PCNA)及凋亡蛋白質酶原-3(pro-caspase-3)兩者表現量皆減少,且活化的凋亡蛋白質酶-3提升(active-caspase-3)(p<0.001,圖9B及9C)。
Pkd1
L3/L3
;Ngal
Tg/Tg
小鼠中哺乳動物雷帕霉素靶蛋白質在腎臟的表現量隨著Akt及S6激酶訊息傳導降低而減少
所有多囊腎疾病小鼠相較於其個別對應之同窩非多囊腎疾病之對照組小鼠,mTOR及p-mTOR之表現量皆增加(圖10A)。此外,Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠比起Pkd1 L3/L3 小鼠及Pkd1 L3/L3 ;Ngal -/-小鼠,其mTOR及p-mTOR之表現量是最低的(p<0.001,圖10B-10D)。mTOR的主要上游調控因子(Akt)及下游標的核醣體蛋白質S6激酶(S6K)與細胞增殖有關;利用西方墨點分析(圖11A)磷酸化Akt(p-Akt)及磷酸化S6K(p-S6K)之表現量,結果證明Pkd1 L3/L3 ;Ngal Tg/Tg 小鼠比起其他兩種多囊腎疾病小鼠更具顯著性地降低(p<0.001,圖11B及11C)。
上皮生長因子受體(EGFR)及磷酸化EGFR(p-EGFR)在Pkd1
L3/L3
;Ngal
Tg/Tg
小鼠腎臟的表現量
EGFR及p-EGFR之高度表現及頂端錯位(apical mislocation)會促進囊泡生長,而抑制EGFR酪胺酸激酶活性可減弱多囊腎疾病的發展(Orellana SA,et al.,Kidney Int, 47:490-499,1995;Du J,et al.,
Am J Physiol, 269:C487-495,1995;Sweeney WE,et al.,Kidney Int, 57:33-40,2000;Torres VE,et al.,Kidney Int, 64:1573-1579,2003)。本發明西方墨點分析(圖12A)證明:相較於其個別對應之同窩非多囊腎疾病之對照組小鼠,三種多囊腎疾病小鼠的EGFR及p-EGFR(Tyr1068)表現量皆提升(p<0.001,圖12B及12C),但三種多囊腎疾病小鼠的p-EGFR/EGFR未具有顯著差異(圖12D)。
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Claims (6)
- 一種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)用於製造藥物之用途,該NGAL係用於預防或治療多囊腎疾病(Polycystic Kidney Disease,PKD)。
- 如申請專利範圍第1項所述之用途,其中該多囊腎疾病係常染色體顯性多囊腎疾病(Autosomal dominant Polycystic Kidney Disease,ADPKD)。
- 如申請專利範圍第1或2項所述之用途,其中該預防或治療係指抑制囊泡擴大、減緩間質性纖維化、增加腎細胞細胞凋亡速度、減緩腎細胞增殖速度或其組合。
- 如申請專利範圍第1或2項所述之用途,其中該NGAL係經由增加活化細胞凋亡蛋白質-3(active-caspase-3),以預防或治療多囊腎疾病。
- 如申請專利範圍第1或2項所述之用途,其中該NGAL係經由減少下列因子之表達;α-平滑肌肌動蛋白質(α-SMA)、缺氧誘導因子-1α(hypoxia-inducible factor 1-α,HIF-1α)、凋亡蛋白質酶原-3(pro-caspase 3)、增殖細胞核抗原(proliferating cell nuclear antigen,PCNA)、Akt、哺乳動物雷帕霉素靶蛋白質(mammalian target of rapamycin,mTOR)、S6激酶或其組合,以預防或治療多囊腎疾病。
- 如申請專利範圍第1或2項所述之用途,其中該NGAL具有劑量依賴性(dose-dependent)。
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US15/220,687 US10307461B2 (en) | 2015-12-23 | 2016-07-27 | Method of preventing polycystic kidney disease and PKD animal model with exogenous neutrophil gelatinase-associated lipocalin |
EP16204110.7A EP3184116B1 (en) | 2015-12-23 | 2016-12-14 | Neutrophil gelatinase-associated lipocalin for use in prevention or treatment of polycystic kidney disease |
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