200804804 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種利用偵測nephronectin之蛋白質或 核苷酸以篩選與急性腎小管壞死有關之急性腎衰竭。 【先前技術】 急性腎衰竭(Acute renal failure ; ARF)在臨床腎臟學上 是僅次於缺血性損傷以及壞死性損傷最普遍的臨床症狀， 並容易造成咼死亡率，而僅能進行支持性治療[1-3]。因此 臣品床上ARF的早期診斷與迅速治療相當重要。 急性腎小管壞死（Acute tubular necrosis ; ATN)是造 成急性腎衰竭原因之一，會造成腎小管上皮細胞大量脫落 與損失[4,5] ’仍附著於基底層的細胞會去分化，並延伸填補 上皮細胞的間隙，並再極化以及重新分化以重建腎小管上 皮組織之構造與功能[6,7]。在修復與再生過程中，細胞外基 質以及其整合素（integrin)受器有顯著改變[8-10]。細胞間 作用主要藉由細胞表面受器整合素家族進行[U]。整合素為 一龐大的異二聚體受器家族，對於發育相當重要，與癒合、 免疫、腫瘤移轉有關[12，13]。整合素α8β1近來被發現在腎 臟型態上扮演重要角色[14]。在α8β1同合子突變小鼠，其 最初生長以及輸尿管分支會嚴重受損，纖維蛋白質 (fibronectin )、連接蛋白質（vitronectin)、韋刃黏素 c (tenascin-C)、造骨蛋白質（oste〇p〇ntin)以及 nephronectin 皆 為整合素α8β1之配子(ligand)。200804804 IX. INSTRUCTIONS OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to the use of proteins or nucleotides for detecting nephronectin to screen for acute renal failure associated with acute tubular necrosis. [Prior Art] Acute renal failure (ARF) is the most common clinical symptom after ischemic injury and necrotizing injury in clinical nephrology, and it is easy to cause sputum mortality, but only supportive Treatment [1-3]. Therefore, the early diagnosis and rapid treatment of ARF on the bed is very important. Acute tubular necrosis (ATN) is one of the causes of acute renal failure, which causes massive loss and loss of renal tubular epithelial cells. [4,5] 'The cells still attached to the basal layer will dedifferentiate and extend to fill The gap between epithelial cells, and repolarization and re-differentiation to reconstruct the structure and function of renal tubular epithelial tissue [6,7]. During the repair and regeneration process, the extracellular matrix and its integrin receptor are significantly altered [8-10]. Intercellular interactions are primarily carried out by the cell surface receptor integrin family [U]. Integrin is a large family of heterodimeric receptors that are important for development and are involved in healing, immunity, and tumor metastasis [12,13]. Integrin α8β1 has recently been found to play an important role in the kidney type [14]. In the α8β1 homozygous mutant mice, the initial growth and ureteral branch are severely impaired, fibronectin, vitronectin, tenascin-C, osteogenic protein (oste〇p〇) Ntin) and nephronectin are the ligands of integrin α8β1.
Nephronectin又可稱為poem(類骨母前趨細胞上皮生 長因子重複刷緣酶，preoste〇blast epidermal growth factor-like repeat meprin，A5蛋白質以及受體蛋白質酪胺酸 水解酶作用區（domain ))，其被認為是一種黏著分子 5 (本所案號05P0336) 200804804 (adhesion molecule)，具有五個類 egf 作用區（d〇main)、 :個Arg-Gly-Asp(RGD)細胞結合基序（m〇tif)以及一刷緣 酶、A5蛋白質以及文體蛋白質質路胺酸水解酶（mam)作 用區[18，19]。在胚胎令腎臟之發育，狀咖〇獄如mRNA表 現於輸尿官芽體上皮，並作為相關配體以調控在腎臟 型態發生之功能[18,20]。腎臟受損與修復、發育有許多方面 相關，因為其牽涉到上皮細胞的去分化、再生以及再分化。 硝酸鈾誘發急性腎小管壞死包含早期的急性腎小管壞 ，以及在復原階段的腎小管上皮細胞再生。因此，尋找確 二月臟再生的標記分子對於分辨急性腎臟衰竭之階段，在 臨床醫學之應用將有相當大地助益。 【發明内容】 ^有鑑於臨床上有使用生物標記分子以檢測腎臟疾病之 而要，本發明提供一用於檢測腎臟疾病之生物標記分子， 其主要目的在於檢測急性腎臟衰竭之狀態，以供臨床上針 對不同狀態進行治療。 本發明之另一目的在於提供一種腎臟疾病之檢測套 組，可從檢體的檢測瞭解腎臟疾病發生狀態，以進行臨床 上的診斷與治療。 本發明之又一目的在於提供一種腎臟疾病之檢測方 法’用以幫助臨床上之腎臟疾病的診斷與治療。 為達上述目的，本發明提供一種用於檢測腎臟疾病之 生物標記分子，其係選自：nephronectin之寡核苷酸序列、 其互補股或其衍生物、其胺基酸序列或其衍生物、其片段、 其變異體、其對應之抗體或其組合物。 本發明較佳係用於檢測急性腎衰竭，係由腎小管壞死 (本所案號05P0336) 6 200804804 症所造成。 在一較佳實施例中，nephronectin寡核苷酸序 SEQ ID NO : 1 ; nephronectin胺基酸序列係為SEq出糸為 2。 本發明另提供一種用於檢測腎臟疾病之生 子，其係選自：SEQIDN0:1所示之寡核苷酸序列、=圮分 股、其衍生物或SEQ ID N0:2所示之胺基酸序或補 物、其片段、其變異體、其對應之抗體或其組合物2衍生 本發明又提供一種腎臟疾病之檢測套組，包含· 標記分子，其係選自於nephronectin之寡核苷酸序=物 補股和衍生物、其胺基酸序列或其衍生物、复片、=互 異體、其組合物或其對應之抗體。 〃又、其變 本發明又提供一種腎臟疾病之檢測方法，係勺人 步驟：（a)提供一檢體；（b)提供一生物標記分子，其^ 3登下列 SEQ ID N0.1所示之券核苦酸序列、其互補股、其彳味、、 SEQ ID N02所示之胺基酸序列或其衍生物、其片&、复= 異體、其對應抗體或其組合物之生物標記分子；（二、夂 生物標記分子與檢體中之_物質錢，前料測物= 選自·· nephronectin之募核苷酸序列、其互補股、其衍^糸 或胺基酸序列或其衍生物、其片段、其變異體、其組I # 及(d)偵測前述步驟(c)中生物標記分子與檢體中之^ : 接觸後之產物。 ' ’、H勿貝 本發明所述之腎臟疾病為急性腎衰竭，其中一實於能 樣係由腎小管壞死症所造成。檢體係包含尿液、病理^刀二怨 於本發明之一貫施態樣中，前述生物標記分子係可進 一步先行固定於基材上，該基材包含，但不限於：免浐八 析盤或生物晶片。本發明可視情況地將檢體中待測物& = 7 (本所案號05P0336) 200804804 一步先行以螢光標記進行標示，以利後續偵測。 在一較佳實施例中，係可於步驟(d)之前增加一利用二 級抗體辨識吸附對應抗體之步驟。步驟(d)中，當偵測胺基 酸序列時，係可利用酵素連結免疫吸附分析（enzyine-linked immunosorbent assay，ELISA)、放射免疫分析（RIA)或免 疫組織化學染色分析（immunohistochemistry )方法。當彳貞測 券核皆酸序列時’係利用反轉錄核酸分子連鎖反應 (reverse-transcriptase polymerase chain reactioin ^ RT-PCR) 或原位雜交反應（7>x s/仏hybridization)。前述步驟(d)的偵 測方法除上述例示外，任何其他熟習該項技術者所熟知的 免疫分析方法皆可使用，無須特別教示。 本發明所述之”變異體”與nephronectin之原胺基酸序列 具有大於80%之序列同一性。本發明所述之，，衍生物，，係指 於前述核苷酸之序列或其互補股之3,端或5,端修飾其他 核普酸序列，使其仍和原序列具有7〇%或以上相似性之寡 核皆酸序列，較佳為具有90%或以上相似性之寡核苦酸序 列。 本發明利用nephronectin之寡核苷酸序列或胺基酸序 列專为子做為生物標記分子，可有效檢測急性腎臟衰竭之 狀態，以供臨床上針對不同病因進行治療。 【實施方式】 本發明係利用nephronectin之寡核苷酸序列或胺基酸 序列等分子做為生物標記分子，用以瞭解腎臟疾病發生狀 態’以進行臨床上的診斷與治療。 以下係提供利用本發明之實施例詳細說明書本發明之 技術及特點，然本實施例並非用以限定本發明，任何熟悉 (本所案號05P0336) 8 200804804 此技藝者’在不脫離本發明之精神和範圍内，當可作各種 之更動與潤飾。 實施例 實驗材料 1·動物模式 石肖酸触誘發急性腎小管壞死（Uranyl-nitrate induced ATN)動物模式： 八週大雌性C57BL/6小鼠（購自於國家動物研究中 心）°所有小鼠接受硝酸鈾U02(N03)2 · 6H2O(10C^g溶於 5%NaHC03)尾靜脈注射。小鼠分別於注射〇,3,7，14天之 後犧牲。荒集小鼠之血液與尿液進行臨床分析，取腎臟 供分子與組織分析。組織分析之樣本根據標準程序以福 馬林(formalin)固定。 局〇卩缺血-再灌注損傷aschemic reperfusi〇n injury)動物 模式： 一此=物模式係以先前技術方式誘發以。 。局部缺血_ 再灌庄扣傷係以雌性Balb/c小鼠g)為對象，在 Ketamine-Xyiazine麻醉後以微血管夹阻塞其腎門（㈣^ 30分麵。當腎臟呈白色時，表示血流已經阻塞， 成。腎臟血流之重建可藉由移除微血管夾 f…^跃问貝鼽之饭手術組（sham operation )之小鼠 2父 微血管夾堵塞以外，處理方式如前述) 以絲描Μ I θ 、， 设局部缺血處理後4,12,24,72小時 以後犧牲小鼠’絲騎_行歷萃取。 (本所案號05Ρ0336) 9 200804804 2.實驗方法 腎功能檢測： 經由眼窩靜脈叢取得血液樣本，離心（3〇〇〇 χ g，1〇分 鐘）後，取出含有血清之上清液後於-70t:下保存。腎功能 藉由測ϊ血清中的肌酸酐（creatinine)與血液尿素氮(bun， Blood Urea Nitrogen)。本實驗所使用之儀器機型為Fuji DRI-CHEM3030(Fuji Photo Film Co. Ltd·，Tokyo, Japan)。 腎臟組織分析： 福馬林固定之腎臟組織以一系列不同濃度酒精脫水 後包埋於石蠟中[13]。切片以後以H&E染色。典型ATN 义才貝日ττ ’月小管細胞壞死時會陷入腎小管板（tubular lamina)。腎小管壞死之定量方式係使用光學顯微鏡。檢 查每一腎臟約100個區塊，根據其組織狀況評分：〇分為 正常組織；1分為腎小管細胞腫脹，刷狀緣(brushb〇rder) *失’細胞核聚集，達1/3腎小管外觀顯示細胞核流失； 2分為1/3-2/3的腎小管外觀顯示細胞核流失；3分為超過 2/3的腎小管外觀顯示細胞核流失。所有ι〇〇格區域分數 加總最高為300分。腎小管細胞再生的證據為平整的上 皮細胞且細胞核之染色質增加（hyperchromatic)且產生 有絲分裂[14]。腎小管細胞再生之定量方式係使用光學顯 微鏡。檢查每一腎臟約1〇〇個區塊，根據其組織狀況評 刀〇刀為正#組織，1分為平整的上皮細胞具有細胞核 之染色質增加與有絲分裂現象；2分為1/3-2/3的腎小管 外觀顯不染色質增加與有絲分裂現象；3分為超過2/3的 腎小管外觀顯示染色質增加與有絲分裂現象。所有1〇〇 格區域分數加總最高為300分。 (本所案號05P0336) 10 200804804 反轉錄聚合酶鏈反應（reverse transcriptase- polymerase chain reaction，以下簡稱 RT_PCR): 以 Trizol(Invitrogen Corporation, Carlsbad,CA)萃取 腎臟之全部RNA，接著使用1.5pg的全部RNA進行一單一 次數的RT反應以合成cDNA之第一股。反應混合液包含 0.9pL 01igo(dT)12-18 引子、l.OmM dNTPs、IX 第一股緩衝 液、0.4mM DTT、80單位RNaseout核糖核酸水解酶抑制劑 以及 300 單位的 superscript II RNase H(Invitrogen Corporatoin，Carlsbad，CA)。以 lpLRT 反應產物做為模版， 加入0·4μΜ具基因特異性之弓|子5TTTTTTTTTTTTTTT、IX PC2 缓衝液、0.25mM dNTPs 以及 1.5 單位的 KlenTaq DNA 聚合酶(Ab Peptides Inc·，St· Louis, M0)。其增幅條件為 94 °C2分鐘、94°C30秒、58°C45秒以及72°C1分鐘（25循環）， 最終再以72°C 10鐘結束反應。β肌動蛋白質（actin)與其他目 標基因產物以1%洋菜膠進行電泳分離，並以溴化乙菲錠 (ethidium bromide, EtBr)染色之。即時聚合酶鏈反應 (real-time PCR)係使用ABI Prism 7700序列偵測系統 (Applied Biosystems，Foster City，CA，USA)。所有探針與引 子係使用 Assay-on-Demand Gene expression products (Applied Biosystems)。即時聚合酶鏈反應係使用lOpL的 cDNA、12·5μί TaqMan Universal PCR Master Mix(Applied Biosystems)、1.25pL專一性探針/引子其序列為 5，GGGAAGGGACAAAGAAGATAG 、 3，AAAGGAACTGGGAGTGTTCTG 混合為 25μί 之混合液。循 環加熱器之反應條件為：50°C 2分鐘（1個循環），95°C 10 分鐘（1個循環），95°C 15秒（40個循環）；60°C 1分鐘。 (本所案號05KB36) 11 200804804 增幅之標準化係以β肌動蛋白質使用2-△△以進行之。 西方墨點法： 每一樣本於12% SDS-PAGE進行電泳，接著將膠體 以轉印至硝化纖維膜（nitrocellulose membrane)上，浸置於 填充緩衝液（blocking buffer ; TBS與5%脫脂奶粉），之 後以TB S T洗漪:三次並浸泡於兔子抗nephronectin，抗體 (Trans Genic Inc·，Kumamoto Japan) 4°C 隔夜。接著將硝化纖 維膜洗滌三次以後與過氧化酶-綴合的山羊抗-兔子抗體 (Pierce, I11，USA)於室溫含浸1小時。膜洗滌三次以後以化 學螢光試劑（Perin Elmer Life 5(^1^6，]\4八，118八)偵測抗體 並以X光片顯影之。 免疫組織染色： 免疫組織染色係使用於福馬林固定石蠟包埋的組 織，以卵白素一生物素（avidin-biotin)免疫過氧化酶方 法偵測之[15]。所使用之抗體包括兔子抗nephronectin及兔 子抗 PCNA(proliferating cell nuclear antigen)(Santa Cruz Biotechnology Inc·，Santa Cruz, CA)。石蠟由組織移除以 後接著再水合(rehydration)。内生性過氧化酶活性被抑制 以後，組織切片與1% w/v BSA(in PBS)缓衝液浸泡一小 時。接著組織切片浸泡於1:100稀釋的兔子多株抗 nephronectin抗體（in PBS)。浸泡於含生物素之二級抗體 後(DAKO，Glostrup，Denmark)，組織切片以卵白素一生物 素免疫過氧化酶複合物（DAKO，Glostrup，Denmark)處理 之。組織切片以蘇木精（Hematoxylin )作為對比染劑 (Counterstain )，並以 3’3-二胺基聯苯胺 12 (本所案號05P0336) 200804804 (3,3’-diaminobenzidine )為染核劑。雙染色之後，利用上 述之 ABC (avidin-biotin -peroxidase complex)使玻片浸 泡於弟一抗體’浸泡於檸檬酸緩衝液（Citrate buffer)並 加以微波。接著玻片浸泡於第二抗體一小時候，鹼性石粦 酸酶（alkaline phosphatase)呈色方式如前述[16]。 nephronectin 以及 PCNA ( (proliferating cell nuclear antigen))之定量係以高能量光學顯微鏡進行。每一區域 隨機選取皮質20個地方，初步檢視腎小管區域正染色的 狀況。比例分數代表陽性反應腎小管細胞比例（1=0-20% , 2=20-40% ’3=40-60% ’4=60-80°/。，5=80-100% )，強度分數 代表腎小管細胞平均染色強度（〇=未染色；1 =弱色；2= 中等；3=強色），比例分數與強度分數加總為正染色的總 量0 免疫電子顯微研究： 樣本以4%之三聚甲酸（paraformaldehyde)與0.5% 戊一备浴於PBS，ρΗ7.4固定之後’根據一般電子顯微鏡 切片處理方式將樣本最後包埋於LR White包埋樹脂中 [15 ]。接者進行超薄切片處理並置於錄網格上。並使用初 級抗體（兔子抗 nephronectin; 1:100; Santa Cruz,CA)與二 級才几體（1··40;金標記，British Biocell International MCardiff，UK)處理。l〇nm金顆粒以電子顯微鏡觀察。 原位雜交技術 3μιη石蠟切片固定於玻片上儲存於密封盒子70°c。 基因核酸探子（riboprobe )以含有nephronectin之cDNA 的pGEMT-EASY質體產生反義RNA。試管内轉錄係使用 (本所案號05P0336) 13 200804804 一般商業化套組進行。原位雜交進行時，Dig標記之 mRNA 以雜交緩衝液（含有 2mM EDTA，pH7.5;20mM Tris， ρΗ7·5 ; 0.6MNaCl ; 2XDenhard，s 溶液；20%葡萄糖銨； O.lmg/mltRNA 以及 〇·2Μ 硫基還原劑（dithiothreitol))稀 釋100倍。去石蠟之後，腎臟切片以2(^g/ml溶於PBS 的蛋白質酶K處理20分鐘。接著切片以0.25%醋酸酐 (acetic anhydride)溶於 〇·ιμ 三乙醇胺（Triethanolamine)進 行乙酿化反應。取約25-50μ1雜交混合液置於每個切片上 並蓋上矽化處理之蓋玻片。雜交反應於濕潤之盒子約42 °C反應16小時。接著蓋玻片移除後以lx ssc (saiine sodium citrate)室溫下洗務一次之後，以〇 2 SSC於室溫下 洗滌10分鐘。之後玻片以〇·〇5χ SSC洗滌室溫下10分 鐘，接著以0.025Χ SSC於37t：洗滌30分鐘。玻片以順 丁烯緩衝液(0·1 M maleic acid，ρΗ7·5; 0.15M NaCl)室溫 /閏/照1为4里以後’以IX填充緩充液（blocking buffer) 反應60分鐘，接著與基因核酸探子（1:2〇⑴於4yc反應隔 夜。之後以順丁浠缓衝液洗滌後於黑暗中以NBT/BCIP 溶液沖洗。雙染色以後，切片與檸檬酸鹽一起微波，並 且如前述文獻所示進行IHC處理。 九知例二·、以本發明之生物標記分子nephronectin檢測急 性腎小營瓌死 在硝酸鈾誘發急性腎小管壞死（Uranyl -nitrate induced ATN)動物模式中，測量不同時間點下之小鼠血 清中的肌酸酐（creatinine)與血液尿素氮（bun, Blood Urea Nitrogen)。根據第一圖A顯示，第三天時血清中的 肌酸酐快速升高，之後肌酸酐的量逐漸下降與控制組 14 (本所案號05P0336) 200804804 (0·29土0.19mg/dL )相當。根據第一圖B顯示第三天之 BUN值升至最高之後下降至與控制組相當。 進一步以顯微鏡檢視梢酸轴誘發急性腎小管壞死動 物模式中’弟二圖為腎臟於注射0,3,7,14天之切片，正常 對照組參照第二圖A。在注射第三天時腎小管有顯著壞死 現象，細胞腫脹、刷狀邊緣消失且細胞核有濃縮現象（參 照第二圖B，E)。而在第七天時有較嚴重的擴散現象（參照 第二圖C，E)，但在第七天顯示平坦的上皮細胞具有細胞 核核濃染（hyperchromatic nuclei)現象顯示腎小管新生。第 十四天時，組織改變較為緩和，且大部分被新生之腎小 管所取代（參照第二圖D，E)。 接著分離硝酸鈾注射後不同時間點之小鼠腎臟 RNA，利用同步定量聚合酶鏈反應（Reai-time pCR)檢 視nephronectin之基因表現狀況。根據第三a圖顯示 nephronectin之RNA表現隨著硝酸鈾誘發急性腎小管壞 死過程而增減，於第三天增加兩倍而在第七天增加五 倍，之後表現量便減少。又根據第三圖B顯示，於局部 缺血-再灌，主^貝傷動物模式下’第三圖B顯示nephronectin 早期表現量升高，之後表現量便減少至正常量。由此結 果可知在鈾誘發急性腎小管壞死以及局部缺血_再灌注損 傷中的去分化與新生過程與nephronectin有關。Nephronectin, also known as poem (preoste〇 blast epidermal growth factor-like repeat meprin, A5 protein and receptor protein tyrosine hydrolase domain), It is considered to be an adhesive molecule 5 (Our Office 05P0336) 200804804 (adhesion molecule) with five classes of egf regions (d〇main), : Arg-Gly-Asp (RGD) cell binding motifs (m 〇tif) and a brush edge enzyme, A5 protein and streptozoal acid aglycol hydrolase (mam) action zone [18,19]. In the embryo, the development of the kidney, such as mRNA, appears in the urinary bud epithelium, and acts as a ligand to regulate the function of the kidney type [18,20]. Renal impairment is associated with many aspects of repair and development because it involves dedifferentiation, regeneration, and re-differentiation of epithelial cells. Acute tubular necrosis induced by uranium nitrate contains early acute tubular failure and tubular epithelial cell regeneration during the recovery phase. Therefore, it is helpful to find the marker molecules that are indeed regenerated in February for the diagnosis of acute kidney failure in clinical medicine. SUMMARY OF THE INVENTION In view of the clinical use of biomarker molecules for detecting kidney diseases, the present invention provides a biomarker molecule for detecting kidney diseases, the main purpose of which is to detect the state of acute renal failure for clinical use. Treatment on different states. Another object of the present invention is to provide a test kit for kidney diseases, which can be used to detect the state of kidney disease from the detection of the specimen for clinical diagnosis and treatment. It is still another object of the present invention to provide a method for detecting kidney disease' to aid in the diagnosis and treatment of clinical kidney diseases. To achieve the above object, the present invention provides a biomarker molecule for detecting a kidney disease, which is selected from the group consisting of: an oligonucleotide sequence of nephronectin, a complementary strand thereof or a derivative thereof, an amino acid sequence thereof or a derivative thereof, A fragment thereof, a variant thereof, a corresponding antibody thereof, or a combination thereof. The present invention is preferably used for the detection of acute renal failure caused by tubular necrosis (Our. No. 05P0336) 6 200804804. In a preferred embodiment, the nephronectin oligonucleotide sequence is SEQ ID NO: 1; the nephronectin amino acid sequence is SEq. The present invention further provides a progeny for detecting a kidney disease, which is selected from the group consisting of: an oligonucleotide sequence represented by SEQ ID NO: 1, a 圮 圮 strand, a derivative thereof, or an amino acid represented by SEQ ID NO: 2. Or a complement, a fragment thereof, a variant thereof, a corresponding antibody thereof or a combination thereof 2 Derivatives The invention further provides a test kit for a kidney disease comprising: a marker molecule selected from the group consisting of an oligonucleotide of nephronectin Order = compound and derivative, amino acid sequence or derivative thereof, multi-sheet, = dissimilar, composition thereof or its corresponding antibody. Further, the present invention provides a method for detecting a kidney disease, which is a step of (a) providing a sample; (b) providing a biomarker molecule, which is represented by the following SEQ ID NO: a biomarker of a nucleus acid sequence, a complementary strand thereof, an astringency thereof, an amino acid sequence represented by SEQ ID NO: 2 or a derivative thereof, a tablet thereof, a complex, a heterologous antibody, a corresponding antibody thereof, or a combination thereof Molecular; (2) 夂 biomarker molecule and _ substance money in the sample, pre-measurement object = nucleotide sequence selected from nephronectin, its complementary strand, its derivative or amino acid sequence or The derivative, the fragment thereof, the variant thereof, the group I # and (d) detect the product of the biomarker molecule in the above step (c) and the contact in the sample: ' ', H Bebeben Institute The kidney disease is acute renal failure, one of which is caused by renal tubular necrosis. The test system contains urine, pathology, and the consistent behavior of the present invention, the aforementioned biomarker molecular system. Further fixed to the substrate, the substrate includes, but is not limited to, a free disk or The present invention can optionally mark the object to be tested in the sample & = 7 (Our. No. 05P0336) 200804804 with a fluorescent mark for subsequent detection. In a preferred embodiment, A step of identifying a corresponding antibody by using a secondary antibody may be added before step (d). In step (d), when detecting the amino acid sequence, an enzyme-linked immunosorbent assay may be used. , ELISA), radioimmunoassay (RIA) or immunohistochemical staining (immunohistochemistry) method. When detecting the nucleus of the nucleus, the reverse-transcriptase polymerase chain reactioin ^ RT- PCR) or in situ hybridization reaction (7>xs/仏hybridization). The detection method of the above step (d) can be used in addition to the above examples, and any other immunoassay method well known to those skilled in the art can be used without special teaching. The "variant" of the present invention has greater than 80% sequence identity with the pro-amino acid sequence of nephronectin. The present invention, derivatives, Means an oligonucleotide sequence which modifies the nucleotide sequence of the nucleotide sequence of the aforementioned nucleotide or its complementary strand at 3, or 5, to have a similarity to the original sequence of 7 % or more. Preferably, the oligonucleotide sequence having 90% or more similarity is used. The present invention utilizes an oligonucleotide sequence of nephronectin or an amino acid sequence as a biomarker molecule, and can effectively detect the state of acute renal failure. For clinical treatment of different causes. [Embodiment] The present invention utilizes a molecule such as an oligonucleotide sequence of nephronectin or an amino acid sequence as a biomarker molecule for understanding the state of occurrence of kidney disease for clinical diagnosis and treatment. The present invention is not limited to the present invention by the detailed description of the embodiments of the present invention. However, the present invention is not intended to limit the present invention, and any one skilled in the art (the present invention, the number of the patent number 05P0336) 8 200804804 In the spirit and scope, when you can make a variety of changes and retouching. EXAMPLES Experimental Materials 1. Animal Patterns Uranyl-nitrate induced ATN Animal Model: Eight-week-old female C57BL/6 mice (purchased from the National Center for Animal Research) ° All mice received Uranium nitrate U02 (N03) 2 · 6H2O (10C ^ g dissolved in 5% NaHC03) was injected intravenously. Mice were sacrificed after 3, 7, and 14 days of injection. The blood and urine of the wild mice were analyzed clinically, and the kidneys were analyzed for molecular and tissue analysis. Samples of tissue analysis were fixed in formalin according to standard procedures. Aschemic reperfusi〇n injury) Animal model: One = the pattern of the disease was induced in a prior art manner. . Ischemia _ Refilling Zhuangchuang is based on female Balb/c mice g). After Ketamine-Xyiazine anesthesia, the renal hilum is blocked with microvascular clamps ((4)^30 min. When the kidney is white, it indicates blood. The flow has been blocked, and the reconstruction of the renal blood flow can be performed by removing the microvascular clamp f...^ 跃 问 小鼠 鼽 手术 s s s s s s s s s s s s s s s s s s s s s s s s s s s s s Trace I θ , and sacrifice the mouse 'silk ride _ calendar extract after 4, 12, 24, 72 hours after ischemia treatment. (Our case number 05Ρ0336) 9 200804804 2. Experimental method Renal function test: Blood samples were taken through the orbital venous plexus, centrifuged (3 〇〇〇χ g, 1 〇 minute), and the serum containing serum was taken out and then 70t: Save under. Renal function by measuring creatinine in serum and blood urea nitrogen (bun, Blood Urea Nitrogen). The instrument model used in this experiment was Fuji DRI-CHEM 3030 (Fuji Photo Film Co. Ltd., Tokyo, Japan). Renal tissue analysis: The formalin-fixed kidney tissue was dehydrated in a series of different concentrations of alcohol and embedded in paraffin [13]. After sectioning, it was stained with H&E. The typical ATN is only able to fall into the tubular lamina when the tubule cells are necrotic. The quantitative method of tubular necrosis is performed using an optical microscope. Check about 100 blocks of each kidney, according to its tissue status score: 〇 is divided into normal tissue; 1 is divided into tubular cell swelling, brushb〇rder * loss 'nucleus aggregation, up to 1/3 renal tubule Appearance showed nuclear loss; 2 1/3-2/3 of the tubular appearance showed nuclear loss; 3 divided into more than 2/3 of the tubular appearance showed nuclear loss. All ι 区域 regional scores add up to a maximum of 300 points. Evidence for tubular cell regeneration is flat epithelial cells with hyperchromaticity in the nucleus and mitosis [14]. The quantitative method of tubular cell regeneration is the use of optical microscopy. Check each kidney about 1 block, according to its tissue condition, the knife is the positive # tissue, 1 divided into flat epithelial cells with nuclear chromatin increase and mitosis; 2 divided into 1/3-2 The /3 tubular appearance showed no increase in chromatin and mitosis; 3 divided into more than 2/3 of the tubular appearance showed an increase in chromatin and mitosis. The total score for all 1 区域 regions is up to 300 points. (Originary Case No. 05P0336) 10 200804804 Reverse transcriptase-polymerase chain reaction (RT_PCR): All RNA of the kidney was extracted with Trizol (Invitrogen Corporation, Carlsbad, CA), followed by 1.5 pg of all RNA is subjected to a single number of RT reactions to synthesize the first strand of cDNA. The reaction mixture contained 0.9 pL of 01igo(dT)12-18 primer, 1.0 mM dNTPs, IX first buffer, 0.4 mM DTT, 80 units of RNaseout ribonuclease inhibitor, and 300 units of superscript II RNase H (Invitrogen) Corporatoin, Carlsbad, CA). Using the lpLRT reaction product as a template, add 0.4 μM gene-specific bow | 5TTTTTTTTTTTTTTT, IX PC2 buffer, 0.25 mM dNTPs, and 1.5 units of KlenTaq DNA polymerase (Ab Peptides Inc., St. Louis, M0) . The growth conditions were 94 ° C for 2 minutes, 94 ° C for 30 seconds, 58 ° C for 45 seconds, and 72 ° C for 1 minute (25 cycles), and finally the reaction was terminated at 72 ° C for 10 minutes.肌actin and other target gene products were electrophoretically separated by 1% acacia and stained with ethidium bromide (EtBr). Real-time PCR was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Assay-on-Demand Gene expression products (Applied Biosystems) were used for all probes and primers. The real-time polymerase chain reaction system used a mixture of lOpL cDNA, 12·5 μί TaqMan Universal PCR Master Mix (Applied Biosystems), 1.25 pL specific probe/primer sequence of 5, GGGAAGGGACAAAGAAGATAG, 3, AAAGGAACTGGGAGTGTTCTG mixed to 25 μί. The reaction conditions of the circulating heater were: 50 ° C for 2 minutes (1 cycle), 95 ° C for 10 minutes (1 cycle), 95 ° C for 15 seconds (40 cycles); 60 ° C for 1 minute. (Our Case No. 05KB36) 11 200804804 The standardization of the increase is carried out by using 2-ΔΔ with β-actin. Western blotting method: Each sample was electrophoresed on a 12% SDS-PAGE, and then the colloid was transferred to a nitrocellulose membrane and immersed in a blocking buffer (TBS and 5% skim milk powder). Then, wash with TB ST: three times and soak in rabbit anti-nephronectin, antibody (Trans Genic Inc., Kumamoto Japan) 4 ° C overnight. The nitrocellulose membrane was then washed three times and then impregnated with a peroxidase-conjugated goat anti-rabbit antibody (Pierce, I11, USA) for 1 hour at room temperature. After the membrane was washed three times, the antibody was detected with a chemical fluorescent reagent (Perin Elmer Life 5 (^1^6,]\4, 1188) and developed with an X-ray film. Immunohistochemical staining: Immunohistochemical staining was used in Fuma. The tissue embedded in paraffin-embedded tissue was detected by avidin-biotin immunoperoxidase method. [15] The antibodies used include rabbit anti-nephronectin and rabbit anti-PCNA (proliferating cell nuclear antigen) ( Santa Cruz Biotechnology Inc., Santa Cruz, CA. Paraffin was removed from the tissue and then rehydrated. After endogenous peroxidase activity was inhibited, tissue sections were plated with 1% w/v BSA (in PBS) buffer. Soak for one hour. Then tissue sections were immersed in a 1:100 dilution of rabbit anti-nephronectin antibody (in PBS). Soaked in biotin-containing secondary antibody (DAKO, Glostrup, Denmark), tissue sectioned with avidin-bio Treatment with a peroxidase complex (DAKO, Glostrup, Denmark). Tissue sections with Hematoxylin as a counterstain (Counterstain) and 3'3-diaminobenzidine 12 (this Case No. 05P0336) 200804804 (3,3'-diaminobenzidine) is a nucleating agent. After double staining, the slide is immersed in the citrate buffer by using the ABC (avidin-biotin-peroxidase complex) described above. Citrate buffer) and microwave. Then slide the immersed in the second antibody for a small time, the alkaline phosphatase coloring method is as described above [16]. Quantification of nephronectin and PCNA (proliferating cell nuclear antigen) The high-energy light microscopy was performed. 20 regions of the cortex were randomly selected from each region, and the positive staining of the renal tubular region was initially examined. The proportional fraction represented the proportion of positive tubular cells (1=0-20%, 2=20-40). % '3=40-60% '4=60-80°/.,5=80-100%), the intensity score represents the average staining intensity of renal tubular cells (〇=unstained; 1=weak color; 2=moderate; 3=strong color), the proportional fraction and the intensity score are added to the total amount of positive staining. 0 Immunoelectron microscopy study: The sample is prepared with 4% paraformaldehyde and 0.5% pentacene in PBS, ρΗ7.4 After fixing 'according to general electron microscopy Finally, slicing processing manner samples were embedded in LR White embedding resin [15]. The receiver performs ultra-thin slicing and is placed on the recording grid. Primary antibodies (rabbit anti-nephronectin; 1:100; Santa Cruz, CA) were treated with a secondary antibody (1·40; gold label, British Biocell International MCardiff, UK). The l〇nm gold particles were observed by an electron microscope. In situ hybridization technique 3 μιη paraffin sections were mounted on slides and stored in sealed boxes at 70 °C. The riboprobe produces antisense RNA from the pGEMT-EASY plastid containing the cDNA of nephronectin. The use of in vitro transcripts (Our Case No. 05P0336) 13 200804804 General commercialization. When in situ hybridization, Dig-labeled mRNA was used as a hybridization buffer (containing 2 mM EDTA, pH 7.5; 20 mM Tris, ρΗ7·5; 0.6 M NaCl; 2X Denhard, s solution; 20% glucose ammonium; O.lmg/ml tRNA and 〇 • 2 Μ dithiothreitol) diluted 100 times. After deparaffinization, the kidney sections were treated with 2 (^g/ml proteinase K in PBS for 20 minutes. Then the sections were treated with 0.25% acetic anhydride dissolved in ethanol·ιμ Triethanolamine for the brewing reaction. Approximately 25-50 μl of the hybridization mixture was placed on each section and covered with a deuterated cover slip. The hybridization reaction was carried out in a humidified box at about 42 ° C for 16 hours. Then the coverslip was removed and then lx ssc ( Saine sodium citrate) After washing at room temperature, wash with 〇2 SSC for 10 minutes at room temperature. The slides were then washed with 〇·〇5χ SSC for 10 minutes at room temperature, followed by 0.025 Χ SSC at 37t: washing 30 Minutes. Slides with butylene buffer (0.1 M maleic acid, ρΗ7·5; 0.15 M NaCl) room temperature / 闰 / 1 for 4 liters later 'filling buffer with IX (reaction buffer) reaction 60 Minutes, followed by reaction with a genetic nucleic acid probe (1:2 〇(1) at 4 yc overnight. After washing with cisplatin buffer, rinse with NBT/BCIP solution in the dark. After double staining, the slices were microwaved with citrate, and IHC processing is performed as shown in the aforementioned documents. Determination of creatinine and blood urea in serum of mice at different time points in the animal model of acute renal tubular necrosis induced by nephronectin Nitrogen (bun, Blood Urea Nitrogen). According to the first panel A, the creatinine in the serum increased rapidly on the third day, and then the amount of creatinine gradually decreased with the control group 14 (Our Office No. 05P0336) 200804804 (0 · 29 soil 0.19mg / dL) equivalent. According to the first figure B, the BUN value on the third day rose to the highest and then fell to the control group. Further microscopic examination of the acid-induced axis of acute tubular necrosis in the animal model The second picture shows the kidneys in the 0, 3, 7 and 14 days of injection. The normal control group refers to the second figure A. On the third day of injection, the renal tubules are significantly necrotic, the cells are swollen, the brush-like edges disappear and the nuclei are concentrated. Phenomenon (see Figure B, E). On the seventh day, there is a more serious diffusion phenomenon (see Figure C, E), but on the seventh day, the flat epithelial cells have nuclear staining ( Hyperchromatic nuclei) showed tubular revascularization. On the fourteenth day, the tissue changes were more moderate and most of them were replaced by new tubules (see Figure D, E). Then separate the uranium nitrate injection at different time points. In mouse kidney RNA, the gene expression of nephronectin was examined by simultaneous quantitative polymerase chain reaction (Reai-time pCR). According to the third graph, the RNA expression of nephronectin increased or decreased with the uranium nitrate-induced acute tubular failure, which was doubled on the third day and increased five-fold on the seventh day, after which the performance decreased. Further, according to the third panel B, in the ischemic-re-irrigation, the main-bead-injury animal mode, the third panel B shows that the early expression of nephronectin is increased, and then the expression amount is reduced to the normal amount. As a result, it is known that dedifferentiation and neonatal processes in uranium-induced acute tubular necrosis and ischemia-reperfusion injury are associated with nephronectin.
進一步以原位雜交技術檢視nephronectin之mRNA 於細胞表現狀況並同時以組織螢光染色檢視nephronecUn 蛋白質在細胞之表現與分佈狀況。蒐集鈾誘發急性腎小 管壞死小鼠第〇,3,7，14天之石蠟包埋腎小管切片進行組 織螢光染色以及原位雜交。實驗結果如第四圖所示，根 據第四圖C，D與第四圖e，F，nephronectin蛋白質與mRNA 15 (本所案號05P0336) 200804804 於皮質之表現在第三天與第七天比第十四天顯著（第四 圖G，H )。Nephronectin之mRNA亦表現於趨質。 nephronectin蛋白質於腎小管皮質顯示nephr〇nectin蛋白 質於第二天至第十四天皆有表現，並於第七天為表現之 高峰。此外，可發現nephronectin蛋白質亦表現於腎小管 髓質，其分佈範圍比控制組廣，鮑氏囊上皮細胞亦於第 三天及第七天有nephronectin蛋白質陽性表現。 由於ATN會造成上皮細胞極性消失以及增生反應， 為瞭解nephronectin蛋白質與前述過程之間的關係，進一 步以免疫組織化學染色技術瞭解腎小管上皮細胞增生的 狀怨以及這兩個蛋白質共同表現的狀態。取鈾誘發急性 月小管壞死小鼠刺激後第〇,3,7，i4天之腎臟以抗 nephronectin以及細胞增生標記分子之抗pcNa抗體雙毕 色，結果如第五圖所示。根據第五圖A，E (深藍色區域） 顯示於第0天表現nephronectin之細胞只有約10% ，並 同時表現PCNA。根據第五圖B顯示刺激後第3天表現 nephronectin之細胞增加，其中大部分細胞尚未表現 PCNA (約75% ) ’到第七天’表現之細胞 激立日’ PCNA之上皮細胞有達95%表現nephronectin (參 照第五圖C，E)，由雙染色方法共同定位顯示，多數新 生腎小管細胞會同時表現這兩種蛋白質。至第十四天 時，由於nephronectin與PCNA被負調節，因此共同表顯 此兩種細胞減少（參照第五圖D，E)。 急性腎衰竭初期之症狀除了包含組織受損傷以外， 尚有細胞極性消失、細胞腫脹無法附著於細胞外基層以 及細胞死亡。進入恢復期時，受損之腎小管會出現細胞 序分化以及細胞分裂之現象，接著腎小管之上皮細胞會 16 (本所案號05P0336) 200804804 被修復並重建細胞極性。第五圖雙染色實驗結果顯示於 ATN弟二天日t PCNA 1¼性細胞亦表現nephr〇nec〖in，只有 少數部分nephronectin陽性細胞表現pcNa。而至第七天 時，多數nephronectin陽性細胞表現pcNA，顯示這些細 胞正在增生當中。而在第十四天（修復晚期），nephr〇nectin 表現下降’，、有少數細胞同時表現nephronectin與 PCNA。此結果顯示，於正在有絲分裂的腎小管細胞加強 表現nephronectin對於調控腎小管壞死的再生相當重 要。Nephronectin被認為在腎臟胚胎早期發育扮演一重要 角色[18,20]。本發明貫施例中觀察到nephronectin在腎臟 功能修復過程中於腎小管上皮細胞再表現可推知兩者之 間的關係’紅合上述之貫驗可知nephronectin之mRNA 與蛋白質在鈾誘發急性腎小管壞死小鼠第七天表現達高 峰，並於腎小管重建後表現急遽下降，由第四圖與第五 圖比較可知nephronectin表現早於pcNA，而腎小管新生 細胞皆會表現 nephronecitn 與 PCNA，顯示 nephronecitn 可藉由整合素α8β1做為一自動分泌反饋迴圈 (automatically secret feedback circuit)以避免近曲腎小 管過度增生。 f施例二、檢測急性腎小管壞死動物模式之尿液中木發 明之生物標記分子nephronectin令吾 接著檢測尿液中是否有nephronectin蛋白質，為避 免蛋白質沈澱，此處以10%非還原SDS-PAGE分析鈾誘 發急性腎小管壞死小鼠後不同天之尿液，結果如第六A 圖所示。以肌酸酐（creatinine)濃度均化SDS-PAGE結 17 (本所案號05P0336) 200804804 果之數值如第六B圖，實驗結果顯示在鈾誘發急性腎小 管壞死小队刺激後第一天，尿液中開始出現 nephronectin，於第二天到高峰並於第九天消失。此一結 果顯示nephronectin蛋白質存於尿液中，可作為急性腎^ 竭生物標記分子並於臨床上作為早期非侵入性檢測。 系示上所述 ，'枣散明係利用nephr〇nectin之募核苷酸序 列;胺基酸㈣等分子做為生物標記好，可有效檢測 ΐ:::小：哀竭為病因的急性腎臟衰竭，可應用於臨 床上針對不同病因進行腎衰竭之治療。 其他實施態樣 本技術領域之人士皆可I:;:前述實施例中，任何熟悉 之精神與範圍内視需要更*^之說明，在不背離本發明 施態樣亦包含在本發明之=飾本發明，因此，其他實 Τ %專利範圍中。 【圖式簡單說明】 第一圖Α係為本發明實 小管壞死動物模式的肌酸酐、"中，硝酸鈾誘發急性腎 #第-圖B係為本發明實j值。 小官壞死動物模式的血液、—中，硝酸鈾誘發急性腎 第二圖Α-D係為本發明^則量值。 腎小管壞死動物模式於〇,3,7^施例一中，硝酸鈾誘發急性 染色之病理組織型態學圖。’，4天後的腎小管切片之η&ε 第二圖Ε係為根據前述 分數所作成數值長條圖。 〜圖A-D之壞死分數與新生 第二圖Α係為本發明實 、也例一中，以硝酸鈾誘發急性 (本所案號05P0336) ^ 18 200804804 腎小管壞死動物模式，利用同步定量聚合酶鏈反應 (Real-time PCR )檢視nephronectin之基因表現狀況。 第三圖B係為本發明實施例一中，以局部缺血-再灌注 損傷(Ischemic reperfusion injury)動物模式，利用同步定量 聚合酶鍵反應（Real-time PCR )檢視nephronectin之基因表 現狀況。 弟四圖A-Η係為本發明實施例一中，以組織免疫化學 染色檢視（A，C，E，G)與原位雜交檢視（B，D，F，H)nephronectin 蛋白質質與mRNA於腎臟中的表現與分佈狀況。 第五A-D圖係為本發明實施例一中，以組織免疫化學 染色技術瞭解腎小管上皮細胞增生的狀態以及nephr〇nectin 和PCNA共同表現的狀態。 第五圖E係為根據前述第五圖A-D之nephronectin與 PCNA表現量分數所作成之數值長條圖。 弟/、圖A係為本發明實施例二中，檢測轴誘發急性腎 小管壞死小鼠尿液中之nephronectin蛋白質含量之西方墨 點法結果。 第六圖B係根據第六圖A之nephronectin蛋白質表現 結果以肌酸酐（creatinine)濃度均化所作成之數值長條圖。 【元件符號說明】 無 19 (本所案號05P0336) 200804804 參考文獻： L ITiadham R, Pascoal M, Bonveulre J¥: renal isifere. NEng! JM&d 334:1448-1460,1996 2. Star RA: Treatment of aaite renal failure. ΚίίΙηψΜί 54:1817-1831, 1998 3, Molitoris BA二 Transiliomng to tiierapy in ischemk acute renal fkihire. /2辦Further, the in situ hybridization technique was used to examine the expression of nephronectin mRNA in the cells and to observe the expression and distribution of nephronecUn protein in the cells by tissue fluorescence staining. The uranium-induced acute tubular necrosis in mice was collected, and the paraffin-embedded renal tubular sections were cultured for 3, 7, and 14 days for tissue fluorescence staining and in situ hybridization. The experimental results are shown in the fourth figure, according to the fourth figure C, D and the fourth figure e, F, nephronectin protein and mRNA 15 (Our Office No. 05P0336) 200804804 in the cortex performance on the third and seventh days Significant on the fourteenth day (figure G, H). Nephronectin mRNA is also expressed in chemokine. The nephronectin protein showed nephr〇nectin protein in the renal cortex from day 2 to day 14, and peaked on the seventh day. In addition, nephronectin protein was also found in the renal medulla, which was more widely distributed than the control group. The baker's capsule epithelial cells also had nephronectin protein positive expression on the third and seventh days. Since ATN causes the polarity of epithelial cells to disappear and proliferative responses, in order to understand the relationship between nephronectin protein and the aforementioned process, immunohistochemical staining techniques are further used to understand the complaints of renal tubular epithelial hyperplasia and the state of common expression of these two proteins. After uranium-induced acute tubular necrosis in mice, the kidneys of the third, seventh, and fourth days of the kidney were treated with anti-nephronectin and anti-pcNa antibodies against cell proliferation markers. The results are shown in Figure 5. According to Figure 5A, E (dark blue region) shows only about 10% of cells expressing nephronectin on day 0, and simultaneously exhibits PCNA. According to Figure 5B, the increase in cells expressing nephronectin on day 3 after stimulation was shown, most of which had not yet expressed PCNA (about 75%) 'to the seventh day' performance of the cell-exciting day' PCNA epithelial cells up to 95% The expression of nephronectin (see Figure 5, C, E), co-localized by the double staining method, shows that most of the neonatal tubular cells will display both proteins simultaneously. By the 14th day, since nephronectin and PCNA were negatively regulated, the two kinds of cells were shown to be reduced (refer to Fig. D, E). In addition to the damage to tissues, the initial symptoms of acute renal failure still have cell polarity disappeared, cell swelling cannot adhere to the extracellular basement, and cell death. Upon entering the recovery phase, the damaged tubules will undergo cell differentiation and cell division, and then the renal tubular epithelial cells will be repaired and reconstituted in the cell polarity 16 (October 05P0336) 200804804. The results of the double staining experiment in Fig. 5 showed that PCNA 11⁄4 sex cells also showed nephr〇nec〗 in the second day of ATN, and only a few nephronectin positive cells showed pcNa. By the seventh day, most nephronectin-positive cells showed pcNA, indicating that these cells are proliferating. On the fourteenth day (late repair), nephr〇nectin showed decreased performance, and a few cells showed both nephronectin and PCNA. This result indicates that the enhancement of nephronectin in the mitotic tubular cells is important for regulating the regeneration of tubular necrosis. Nephronectin is thought to play an important role in the early development of kidney embryos [18,20]. In the present invention, the relationship between nephronectin and renal tubular epithelial cells during renal function repair can be inferred. The relationship between nepronectin mRNA and protein in uranium-induced acute tubular necrosis can be inferred. The mice showed a peak on the seventh day and showed a rapid decline after renal tubular reconstruction. It can be seen from the fourth and fifth images that nephronectin is earlier than pcNA, and the renal tubular neonatal cells will exhibit nephronecitn and PCNA, indicating nephronecitn. Integrin α8β1 is used as an automatic secret feedback circuit to avoid hyperplasia of proximal tubules. f Example 2: Detection of acute tubular necrosis in animal models of urine. The biomarker molecule nephronectin in the invention allows me to detect the presence of nephronectin protein in the urine. To avoid protein precipitation, a 10% non-reducing SDS-PAGE analysis is performed here. The urine of different days after uranium-induced acute tubular necrosis in mice, the results are shown in Figure 6A. Homogenization of creatinine concentration SDS-PAGE knot 17 (Our Case No. 05P0336) 200804804 The value of the fruit is shown in Figure B, and the results show that the urine on the first day after stimulation of the uranium-induced acute tubular necrosis team, urine Nephronectin began to appear in the middle, peaked on the second day and disappeared on the ninth day. This result shows that nephronectin protein is present in the urine and can be used as an acute renal biomarker molecule and clinically as an early non-invasive test. According to the above description, the jujube-dissolving system utilizes the nucleotide sequence of nephr〇nectin; the amino acid (tetra) and other molecules are used as biomarkers, which can effectively detect ΐ:::small: acute kidneys with laxification as the cause Depletion can be applied to the clinical treatment of renal failure for different causes. Any other person skilled in the art of the prior art can use the following description: The present invention, therefore, is in the scope of other patents. [Simple description of the diagram] The first figure is the creatinine of the small tube necrotic animal model of the present invention, and the uranium nitrate-induced acute kidney #第图B is the actual j value of the present invention. The blood of the small necrotic animal model, the middle, the uranium nitrate induced acute kidney. The second figure Α-D is the value of the invention. The animal model of renal tubular necrosis was found in 〇, 3, 7^, and the pathological histological pattern of acute staining induced by uranium nitrate. ', η & ε of the tubular section after 4 days is a bar graph of the numerical value based on the above score. ~ The necrotic fraction of AD and the second map of the newborn are the present invention, also in the first example, with uranium nitrate induced acute (Our Office No. 05P0336) ^ 18 200804804 Kidney tubular necrosis animal model, using synchronous quantitative polymerase chain The gene expression status of nephronectin was examined by Real-time PCR. Figure 3B is a diagram showing the gene expression of nephronectin by the simultaneous quantitative polymerase chain reaction (Real-time PCR) in an ischemic reperfusion injury animal model according to the first embodiment of the present invention. Figure A-Η is the first embodiment of the present invention, using tissue immunochemical staining (A, C, E, G) and in situ hybridization (B, D, F, H) nephronectin protein and mRNA The performance and distribution of the kidneys. The fifth A-D diagram is the first embodiment of the present invention, and the state of renal tubular epithelial cell proliferation and the state of co-expressed by nephr〇nectin and PCNA are understood by tissue immunochemical staining technique. Figure 5E is a bar graph of the values of the nephronectin and PCNA performance scores according to the aforementioned fifth panel A-D. The figure is the western blot method for detecting the protein content of nephronectin in the urine of mice with acute tubular necrosis detected in the second embodiment of the present invention. Figure 6B is a bar graph of the numerical results obtained by homogenizing creatinine concentration according to the protein expression of nephronectin in Figure 6A. [Explanation of component symbols] None 19 (Our Case No. 05P0336) 200804804 References: L ITiadham R, Pascoal M, Bonveulre J¥: renal isifere. NEng! JM&d 334:1448-1460,1996 2. Star RA: Treatment Of aaite renal failure. ΚίίΙηψΜί 54:1817-1831, 1998 3, Molitoris BA II Transiliomng to tiierapy in ischemk acute renal fkihire.
Nephivl 14:265^267,2003 ^ ' 4. Raciisen LC;; Frnish BA, Li IcX, et al: Dissociatioa of tubular cell detadiment md tubular cell death m clinical znd e^enmeiital Maaiie tubular necrosisfS. Lab Invest 64:546-556,1991 5· Racmeii LC: Epidbelial. cell sbedcfing in acute renal ΐϋίυτγ. Cim Exp Phmw>acoi 25:273-275,199S ^ ' 6. Edeklein CL. Ling BL Schrter RW: Tht nsture of renal cdl iiyurv. Kidnm M 51:1341-1351,1997 — 备 7· Fish EML MoMtoris BA: Afteratioos m epithelial polarily and rfie pathogenesis of disease N Engl J Med 330:15Z0A5BSr 1994 5, Μώ Ss Bicfaett l^cholson ML: Molecidar changes in extmodblar matrix tttmover after renal ischaemta-reper&siofi injury, Sr JSurg 87:11SS-1192? 2000 9. Zuk A, Bonveotre JVS D, ei al: Poiarit}·; mtegriix and extracdlukr matrix dynamics in the postischemic mt kidoey. Am I Physio! 275:C711 -731,1998 10. Zuk Ar Hay ED: Expression of beta 1 integrins changes during transfon'Batioii of mim lens epithelium lo mesenchyme in collagen gels. Dm? I)}m 201:37S-393, 1994 ' ^ * 11.. Brown NH: Cdtl-cd! adhesion \ia the ECM: iolegrm geneiks in fly and womi· Matrix Btoi 19:191-201,2000 12 . Hynes RD: Targeted mDiations in cell adhesion genes: what have we leaned &om ihmtl Biol 180:402-412,19% 13. Eassler 1L： Georges-L^ouesse E9 Hirsch E: Genetk analyses of inte^in fimctioa in mice. CkmH Opin Ceil 3io! 8:641-646^ 1996 14. Muller U, Wang D? Denda S5 etaL· Mlegrin alpliaSbetal is critically mipoitant for epiflieliai-nies^nclivnial iiiteractioiis Airing kidaey morphogenesis. Ceil 88:603-613, 1997 * ^ 15. Scimapp Ovt Hatch Rmnos DI%C et al: Hie hmnan integriii alpha 8 beta I ftmctioas as a receptor for toiascin, fibranectiiL. and vitronectin. J Bio! Chem 270:23196-23202,1995 16. Denda S? Reichardt UF? Muller U: Ickntification of osteopontia as a novel ligand iot the integrm alphaB betal and poteiiiai roles for this integrb-ligand mteractioo m kidney morphogenesis. Mo!Btoi Cell 9:1425-1435,1998 17. 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