富含半胱氨酸61蛋白在急性與慢性腎臟病角色的探討

Abstract

全球的末期腎臟病發生率與盛行率仍然持續增加，其中的一個重要原因是，即使以目前最完善的治療，仍無法完全阻止慢性腎臟病的病程進展。動物實驗與臨床研究都發現，不論原發的病因為何，進行性腎臟病最後都會進展為相似的病理機轉，亦即慢性的腎小管間質纖維化。此外，近來的研究也顯示，急性腎損傷以後腎臟無法完全修復，可能會導致持續性的病理變化，並且演變為慢性腎臟病。為了進一步發展更完善的腎臟疾病治療，需要進行更深入的研究，來了解腎小管間質纖維化與急性腎損傷轉換為慢性腎臟病之詳細病理機轉。 富含半胱氨酸61蛋白（Cysteine-rich protein 61，Cyr61）是一個與細胞外間質密切相關的分泌性蛋白質，具有多樣的功能。目前已知Cyr61參與了控制細胞循環、改造細胞外間質、促進血管生成、以及調控發炎反應。過去的臨床研究與動物實驗曾報告過，Cyr61會在正常與疾病狀態的腎臟中表現。然而，目前仍不清楚Cyr61在腎臟疾病中的角色。吾人的研究假說為Cyr61在急性與慢性腎臟疾病病理機轉中具有關鍵性的功能，目的在於探究Cyr61在慢性進行性腎臟纖維化的致病角色，以及探究Cyr61在急性腎臟缺血再灌流損傷後的致病角色。 第一部分吾人利用單側輸尿管阻塞動物模式，探究Cyr61在進行性腎臟纖維化中之表現。相對於偽病組而言，輸尿管阻塞後第一天腎臟的Cyr61基因表現與蛋白質產生量就顯著地增高，並且持續到第十天。腎臟切片的組織免疫染色檢查，發現Cyr61蛋白質主要的表現位置在腎小管上皮細胞。而在細胞實驗中，我們發現transforming growth factor-β1（TGF-β1）可以刺激近端腎小管上皮細胞表現Cyr61。在單側輸尿管阻塞前給予小鼠注射抗TGF-β單株抗體，則可顯著地將術後第1天腎臟Cyr61的過度表現給壓抑下來，此結果支持了細胞實驗的發現。此外，在時序性的研究中，吾人觀察到單側輸尿管阻塞後monocyte chemoattractant protein-1（MCP-1）基因的增加表現緊跟在Cyr61之後，若以anti-Cyr61抗體治療單側輸尿管阻塞的小鼠，可顯著地減少腎臟的MCP-1基因表現，以及阻塞後第四天腎臟中巨噬細胞的浸潤。體外實驗以合成的Cyr61蛋白質處理近端腎小管上皮細胞，可刺激細胞表現MCP-1。這些結果共同揭示了腎小管上皮細胞中TGF-β1 → Cyr61 → MCP-1作用軸的機轉，也顯示出Cyr61具有促進發炎反應的功能。最後，接受了anti-Cyr61抗體治療的單側輸尿管阻塞小鼠，術後第四天腎臟的collagen type 1-α1（Col 1-α1）與α-smooth muscle actin (α-SMA)基因表現量減輕，picrosirius red染色下膠原纖維的組織累積減少，而且α-SMA蛋白質產生量也下降。然而，這個阻斷Cyr61帶來的抗纖維化效應並無法持續到疾病的後期。 第二部分吾人利用腎臟缺血再灌流損傷動物模式，來探究Cyr61在急性腎損傷後的表現。在嚴重單側腎臟缺血再灌流損傷後，腎臟的Cyr61基因表現自手術後第1天起就明顯地增加，直到第14天仍持續高於偽病組。組織免疫螢光染色發現Cyr61主要表現在近端腎小管上皮細胞，而遠端腎小管並無顯著表現。細胞實驗將近端腎小管上皮細胞培養於缺氧/再復氧的狀況下，的確也會增加Cyr61基因表現。以合成的Cyr61蛋白質處理近端腎小管上皮細胞，可觀察到MCP-1與interleukin（IL）-6的基因表現增加，顯示出Cyr61的促發炎功能。再者，吾人以anti-Cyr61抗體治療單側腎臟缺血再灌流損傷的小鼠，術後第七天與第十四天腎臟的MCP-1、IL-6、IL-1β、macrophage inflammatory protein-2基因表現明顯受到抑制，腎臟中巨噬細胞的浸潤也顯著減少。在術後第十四天，anti-Cyr61抗體治療也減輕了腎臟中Col 1-α1、Col 3-α1、TGF-β1、與plasminogen activator inhibitor-1的基因表現，減少picrosirius red染色下膠原纖維的組織累積，以及降低α-SMA蛋白質產生量。同時，我們發現anti-Cyr61抗體治療也可減輕損傷後腎臟的腎小管周圍微血管循環稀薄現象。這些結果顯示，即時地阻斷受傷後的近端腎小管上皮細胞所分泌的促發炎訊息（例如Cyr61），可以減輕急性腎損傷後病理變化所導致的腎小管間質纖維化。此外，我們的臨床研究發現，急性腎損傷病患尿液中，Cyr61蛋白質的排出量顯著增加，印證了基礎實驗的發現。至於Cyr61是否能發展成為一個實用的急性腎損傷生物標誌，以及是否急性腎損傷病患尿液的Cyr61排出量能用來預測日後產生慢性腎臟病，仍需要後續進行臨床研究來加以探究。 總結本研究的結果，吾人的動物實驗發現在單側輸尿管阻塞後透過TGF-β1的刺激造成Cyr61快速地在腎小管上皮細胞增加表現，也觀察到在急性腎臟缺血再灌流損傷後近端腎小管上皮細胞的Cyr61活化。這些結果在細胞實驗與臨床研究中都獲得印證。Cyr61在急性缺血性腎臟病與進行性腎臟纖維化的病程中，主要的功能是促進發炎反應，而非直接影響纖維化或血管生成。Cyr61是一個關鍵性的因子，聯繫了上皮細胞的傷害、發炎反應、微循環稀薄現象、以及腎臟纖維化。這些研究結果擴展了吾人對進行性腎臟纖維化與急性腎損傷轉換為慢性腎臟病之病理機轉的了解，符合臨床上觀察到的現象。未來還需要繼續探究Cyr61在不同疾病期程中與微環境的交互作用，對不同腎臟細胞造成的不同作用，以及對巨噬細胞極化方向的影響，以期能夠針對Cyr61的功能與訊息傳遞途徑，找出治療與預防慢性腎臟病的目標。

End-stage renal disease is still increasingly prevalent. One of the reasons for this phenomenon is the imperfect renoprotective therapy for chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. Furthermore, emerging data have suggested that acute kidney injury (AKI) is often incompletely repaired and can lead to CKD. Understandings the exact cellular mechanisms of tubulointerstitial fibrosis and AKI-to-CKD transition, and identifying potential modifiable factors warrant further studies. Cysteine-rich protein 61 (Cyr61) is a secreted matrix-associated protein that participates in cell cycle control, cell matrix remodeling, angiogenesis, and inflammation. Animal and human studies have demonstrated Cyr61 expression in normal and diseased kidneys. However, the role of Cyr61 in renal disease remains largely unknown. We hypothesized that Cyr61 contributes to the pathogenesis of acute and chronic kidney diseases. This study aimed to understand the pathogenetic role of Cyr61 in progressive renal fibrosis and ischemic renal injury. First, we investigated the role of Cyr61 in progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) surgery in mice. The expression of Cyr61 transcripts and proteins in the obstructed kidneys were increased from day 1 and remained high until day 10 after surgery. Immunohistochemistry indicated that Cyr61 was expressed mainly in renal tubular epithelial cells. The upregulated Cyr61 in UUO kidneys was reduced in mice treated with pan-transforming growth factor-β1 (TGF-β1) antibody. The role of TGF-β1 in tubular Cyr61 upregulation after obstructive kidney injury was further supported by experiments showing that TGF-β1 stimulated Cyr61 expression in cultured tubular epithelial cells. Notably, the upregulation of Cyr61 in UUO kidneys was followed by a marked increase in monocyte chemoattractant protein 1 (MCP-1) transcripts and macrophage infiltration, which were attenuated in mice treated with anti-Cyr61 antibodies. This proinflammatory property of Cyr61 in inducing MCP-1 expression was further confirmed in tubular epithelial cells cultured with recombinant Cyr61 protein. These data provided evidence supporting the TGF-β1 → Cyr61 → MCP-1 axis. The anti-Cyr61 antibody in UUO mice also reduced the levels of collagen type 1-α1 transcripts, collagen fibril accumulation evaluated by picrosirius red staining, and the levels of α-smooth muscle actin (α-SMA) transcripts and proteins on day 4 after surgery; however, the antifibrotic effect was not sustained when the UUO kidneys progressed further. Second, we explored the expression of Cyr61 after unilateral kidney ischemia-reperfusion injury (IRI) in mice. After IRI, increased expression of Cyr61 was detected, predominately in the proximal tubular epithelium. This was confirmed by in vitro experiments, which showed that hypoxia-reoxygenation stimulates Cyr61 expression in cultured proximal tubular epithelial cells. The proinflammatory property of Cyr61 was indicated by its ability to upregulate MCP-1 and interleukin (IL)-6. Notably, treating mice with an anti-Cyr61 antibody attenuated the upregulation of kidney MCP-1, IL-6, IL-1β, macrophage inflammatory protein-2, and reduced the infiltration of F4/80-positive macrophages at day 7 and 14 after IRI. In addition, blocking Cyr61 reduced the mRNA expression of collagen, TGF-β1, and plasminogen activator inhibitor-1, the degree of collagen fibril accumulation, as evaluated by picrosirius red staining, and the levels of α-SMA proteins by day 14. Concurrently in the treated group, peritubular microvascular density was more preserved at day 14. These results suggested that targeting proximal proinflammatory signaling events, such as Cyr61, potentially ameliorates pathological processes that lead to tubulointerstitial fibrosis. Additionally, we found elevated urinary Cyr61 excretion in patients with AKI. Whether Cyr61 is a practical biomarker for AKI, and whether its level in urine reflects progressive kidney injury deserve further large-scale clinical studies. In conclusion, we showed that, through TGF-β1 stimulation, there was an early and continuous upregulation of Cyr61 in renal tubular epithelial cells during the course of kidney fibrosis after UUO. We also demonstrated Cyr61 upregulation after severe kidney IRI in vivo, supported by in vitro experiments, and further validated by examining clinical urine samples from patients with AKI. The major role Cyr61 plays in acute ischemic kidney injury and chronic progressive kidney fibrosis is pro-inflammation, rather than direct fibrogenesis or angiogenesis. Cyr61 is one of the crucial regulators that connect tubular epithelium injury, inflammation, capillary rarefaction, and progressive kidney fibrosis after IRI. These studies expand the knowledge of the mechanisms of progressive kidney fibrosis and AKI-to-CKD transition, and suggest that Cyr61 is a potential therapeutic target.