內皮唾酸蛋白質在慢性腎病之角色

Abstract

不同成因的腎臟疾病最終都會導致腎纖維化，它是器官衰竭的預兆，並且反映出過度的發炎以及不當的器官重組。許多的研究利用動物模型證實抑制纖維產生的訊息路徑能有效減緩腎臟纖維化，而這些研究只有少部分能應用到臨床上。儘管器官纖維化有很高的盛行率，目前仍然沒有很好的治療可以抑制纖維化的進程，因此，研究纖維化的機制可以幫助發展新的治療。 Endosialin 中文為內皮唾酸蛋白質，也被稱作腫瘤內皮標記蛋白1 (TEM1) 或者 CD248，它是第一型的穿膜醣蛋白，主要表現在正常及腫瘤的基質細胞。利用實驗室所建立的 LacZ-KI 小鼠，我們發現在出生後大多數器官都會減少Endosialin的表現，只有腎臟仍持續大量表現。主要細胞型包括腎絲球、周細胞及血管周圍的纖維母細胞。最近文獻指出人類慢性腎病患者的腎臟切片中可測得 endosialin會高量表達在肌纖維母細胞，且表現量與腎功能呈反比。文獻已證實腎臟周細胞及血管周圍的纖維母細胞即為造成腎臟纖維化的肌纖維母細胞，截至目前為止， endosialin 在肌纖維母細胞的角色與影響仍然未知。 本論文主要要探討 endosialin與腎臟纖維化以及巨噬細胞轉型作用的關係，實驗主軸是利用 UUO 模式誘導小鼠產生慢性腎病模擬人類疾病。首先在 UUO處理後，可見 Endosialin 大量表現在腎臟的膠原蛋白製造細胞，失去 Endosialin 會減緩纖維化。此外組織的巨噬細胞數目減少，且M2 巨噬細胞比例也較低。進一步分析發現 LacZ-KI小鼠的巨噬細胞分泌較少的CCL17和CCL22。而在體外共同培養系統誘導 M1/M2 轉型模式下，發現相似結果。顯示 endosialin 可能藉由調控巨噬細胞的 M1 及 M2 轉型而促進纖維化。 進一步欲證明周細胞與巨噬細胞可能藉由endosialin 及galectin-3交互作用而造成周細胞的活化及巨噬細胞的型態改變，本論文使用免疫共沉澱方式證實兩者可相互作用。 本論文結論可知當腎臟受損時，損傷的組織會吸引單核細胞浸潤並活化成M1 巨噬細胞。巨噬細胞也會分泌促纖維化因子造成周細胞活化並大量表現endosialin，巨噬細胞也會分泌 galectin-3 與 endosialin 結合並促進膜外基質產生。同時 endosialin 也會透過未知的機制促使 M1巨噬細胞轉型成 M2 細胞，並且製造更多膜外基質，導致不可逆的纖維化。

Fibrosis is the final common manifestation of a wide variety of kidney diseases and it is a harbinger of organ failure and reflects inadequate resolution of inflammation in response to injury or inappropriate organ remodelling. Many studies targeting key fibrogenic pathways have demonstrated efficacy in mitigating renal fibrosis in experimental models. Only a small fraction of these approaches, however, have been studied in human and even fewer have been successfully translated to clinical use. Despite the prevalence of organ fibrosis, no good therapies directly target the fibrotic process. Thus, understanding the mechanisms of fibrosis in response to injury is pivotal to our development of new therapies to counteract fibrosis. Endosialin, also known as tumor endothelial marker 1 (TEM1) or CD248, is a type I transmembrane glycoprotein that is expressed in stromal cells in normal tissues and cancers. Using lacZ knock-in (+/KI) mice we have shown that Endosialin expression decreases in most organs but increases and persists in postnatal kidneys, specifically in glomerular mesangial cells and perivascular cells. CD248 has also been identified in normal kidney pericytes and perivascular fibroblasts of mice by specific antibody staining. In human chronic kidney disease (CKD), upregulated expression of Endosialin is shown in myofibroblasts, which is linked to renal survival. Many studies including ours have identified pericytes and perivascular fibroblasts as the major source of precursors of scar-producing myofibroblasts during renal fibrosis. The mechanisms underlying the impact of increased endosialin expression in myofibroblasts, namely activated pericytes, on kidney disease progression is not clear now. In this study, we investigated the role of endosialin in fibrosis and macrophage phenotype switch by using UUO model to induce CKD in mice. We found that Endosialin is up-regulated in the collagen producing cells in the kidney of mice, and loss of Endosialin attenuated collagen-induced fibrosis. Furthermore, without Endosialin also decreases the numbers of macrophage. Analysed the gene expression pattern in macrophage, the M2 marker, CCL17 and CCL22 were expressed lower in KI/KI mice. In vitro co-culture of M1/M2 macrophage phenotype-switch assay, similar results were detected in macrophage’s CCL17 and CCL22 when culture with KI/KI cells. These results suggest that endosialin promotes the fibrosis though macrophage phenotype regulation. To further investigate whether the interaction between endosialin and galectin-3 exists and leads to activation of pericytes and pro-fibrotic phenotype switch of recruited macrophages during progressive renal fibrosis. We confirm the interaction between endosialin and galectin-3 by immunoprecipitation. In summary, monocytes would be recruited and activated to become M1 type macrophages by injury signals. Macrophages will secret cytokines to activate preicytes becoming myofibroblasts. Myofibroblast will highly express endosialin and interact with galectin-3 which is secreted by macrophages and consequently enhance collagen production. In addition, endosialin will promote these M1 type macrophages switch to M2 type through unknown mechanism. These changes will activate more myofibroblast and tissue will become more fibrogenic.