內皮唾酸蛋白基因剃除後對缺血再灌流的 內皮唾酸蛋白基因剃除後對缺血再灌流的急性腎損傷之影響

Abstract

即便在現今醫療水準大幅提升的環境下，急性腎損傷 (acute kidney injury；AKI)的發病率、盛行率及死亡率仍高居不下。而至今仍然沒有發展出有效的藥物或治療方式來改善急性腎損傷，因此尋找出有效的藥物或者治療方式是十分迫切的。 根據過去的研究中發現到Endosialin (中文稱內皮唾酸蛋白；又稱Tumor endothelial marker 1；CD248)在健康的成體中，只有腎臟及子宮有大量的表現；在腫瘤生長或者發炎反應時，Endosialin也會被大量的表現出來。先前實驗室在單側輸尿管阻塞的慢性腎臟病模型研究中，發現到將Endosialin剔除後，可以有效地降低腎臟的纖維化，而腎臟內的巨噬細胞數量也會減少；但巨噬細胞在急性發炎所扮演的角色跟慢性發炎又不相同，普遍認為巨噬細胞在急性發炎時對於組織的支持、修復是有正面的幫助，因此我們想看看在急性腎損傷的情況下，Endosialin 剔除後會有什麼樣的影響。 本論文是將7週大的公鼠先施予右側腎臟摘除，兩週後再將左側腎臟做缺血再灌流手術，做為本實驗的急性腎損傷模型，藉此來比較Endosialin基因剔除鼠與C57BL/6小鼠是否有差異性。 實驗的結果顯示，老鼠在受到缺血再灌流的損傷之後，不論是在第二天或第十天，其血液尿素氮和肌酸酐的數值，基因剔除鼠都比一般的小鼠低。而在第二天的PAS stain中，組織的損傷程度也有一樣的趨勢。不過在檢測巨噬細胞相關的RNA表現上，卻沒有看到明顯的變化。所以再利用免疫組織化學染色的方式去看巨噬細胞及嗜中性球浸潤之情形，結果可以看到這兩種免疫細胞的浸潤都沒有差異。接著我們想看看在腎臟細胞的凋亡與增生的情況，於是利用TUNEL assay及Ki67進行免疫螢光染色，結果發現將endosialin剔除後，可以減少細胞的凋亡，並增加細胞的增生。 根據實驗的結果可以知道把endosialin剔除後可以減少IRI造成的腎損傷，並且會促進腎臟細胞的增生。但endosialin的剔除並不會影響到免疫細胞的浸潤，且相關的基因表現並無明顯差異。由於過去的研究指出，endosialin會去影響到血管的新生或者是組織的血液灌流量，我們推測或許是這些原因造成上述的結果，而這部分仍需更多實驗來驗證。 過去臨床報告中指出，發生急性腎損傷的病人即使在治療後恢復到了正常，但在日後轉為慢性腎臟病的機會還是比一般正常人來的高，且AKI的嚴重程度與未來轉變成CKD又有高度的相關性；而本實驗中可以發現到基因剔除鼠在於預防急性腎損傷有良好的效果，未來我們可以利用這個老鼠去觀察其轉為慢性腎病變的機率是否也比正常老鼠來的低，以期許未來可以利用Endosialin的抗體做為一個治療的方式。

Although medical care advances significantly , the incidence, morbidity and mortality of acute kidney injury (AKI) are still high. But there is no promising drug or therapy for AKI treatment, so it is an unmet medical issue to discover effective therapeutics for AKI. According to a previous study, endosialin (also known as tumor endothelial marker-1 or CD248) is only highly expressed in kidney and uterus of a healthy adult; in the tumor growth or inflammation, endosialin will be overexpressed. Previous laboratory studies of chronic kidney disease model of unilateral ureteral obstruction found that the endosialin knockout mice could effectively reduce fibrosis and the number of macrophages within the kidney. Because macrophages play different roles in acute and chronic inflammation, we are intrigued by the effect of endosialin knockout on the AKI. To compare the difference between endosialin knockout mice and wild type mice, we performed right nephrectomy to 7-week-old male mice and induced ischemia-reperfusion injury in the left kidney 2 weeks later, as an AKI model. Our data showed that mice subjected to ischemia- reperfusion injury, the plasma levels of blood urea nitrogen and creatinine were lower in knockout mice at either day 2 or 10 after injury. Periodic-acid Schiff stain showed that extent of tissue damage was less severe in knockout mice, too. In the injured kidneys of knockout mice, moreover, cell apoptosis detected by terminal deoxynucleotidyl transferase dUTP nick end labeling was decreased. In contrast, cell proliferation detected by Ki67 staining was increased. However, we did not detect significant difference in the expression of function-related genes in macrophages using quantitative polymerase chain reaction. The numbers of macrophages and neutrophils infiltration in the injured kidneys detected by immunohistochemistry were not different, either. According to the results, endosialin knockout can attenuate renal injury caused by ischemia-reperfuion and enhance kidney cell proliferation. But the infiltration of macrophages and neutrophils as well as the function-realted gene expression of macrophages in injured kidneys are not changed by enosialin knockout. Because endosialin has been shown to affect angiogenesis and blood perfusion in previous studies, we will explore whether endosialin regulates renal blood flow and microvasculature in heathy and injured kidneys in the future. Evidence has shown that AKI patients, even with functional recovery after supportive care, will experience increased risk for progression to chronic kidney disease. Based on our data showing the potential of endosialin disruption in protection of mice from AKI, we will advance our knowledge for the mechaisms responsible for such a renoprotective effect. Furthermore, we will use this mouse to study whether endosialin knockout can block the progression from AKI to chronic kidney disease.