腎臟病變中糜蛋白酶與第二型血管收縮素轉換酶於病程發展中所扮演角色的探討

Abstract

在腎臟組織的腎素-血管收縮素系統(renin-angiotensin system, RAS)中，血管收縮素轉換酶(angiotensin conversing enzyme, ACE)為將血管收縮素I (angiotensin I, Ang I)轉換成血管收縮素II (angiotensin II, Ang II)的主要途徑，然而在許多腎臟疾病中，已有證據指出chymase會取代ACE將Ang I轉換成Ang II。本研究目的即在於探討chymase在馬兜林酸(aristolochic acid I, AAI)誘發之腎臟病變過程中，RAS中相關酵素扮演之角色。一般的RAS抑制劑，例如ACE抑制劑(ACE inhibitor, ACEI)及Ang II受體阻斷劑(angiotensin II receptor blockers, ARBs)，可以有效地處理早期腎臟疾病之病程發展，但是對於改善腎臟疾病後期的病程發展是有限的。由於先前研究指出，糜蛋白酶(chymase)在某些心血管和腎臟疾病的發展中扮演重要角色，因此本研究將探討chymase抑制劑的給予於AAI誘發腎臟病變的影響。 C57BL/6野生型(Wild-type, WT) 小鼠與第二型血管收縮素轉換酶(angiotensin conversing enzyme II, ACE2)基因剔除(ACE2 KO)小鼠被利用於腎臟病變模型實驗的進行。實驗為於小鼠腹腔注射AAI (累積劑量為45或100 mg/kg小鼠體重)，其經過24小時和2週病情發展期後，犧牲小鼠收集其血液及腎臟組織樣本，分別進行生化、組織病理及分子檢測。在AAI誘發腎臟損傷後，小鼠的體重顯著下降，血清肌酐酸和尿素氮顯著上升，腎臟組織中呈現大量白血球細胞浸潤的炎症反應病徵，且有明顯的腎臟間質纖維化病變。而ACE2 KO小鼠的上述生理與病理病變比WT小鼠更加嚴重。 實驗動物的腎臟組織中、基質金屬蛋白酶(matrix metalloproteinases, MMPs)活性因AAI的處理而改變。在WT小鼠中，AAI處理使腎臟組織中MMP-2活性顯著上升，MMP-9活性則下降，與WT小鼠相比較，ACE2 KO小鼠腎臟組織中的MMPs活性變化更為顯著。在腎臟組織中RAS相關酵素之活性變化，AAI的處理會造成chymase活性的顯著上升，ACE活性下降，而ACE2活性卻顯著上升。在ACE2 KO小鼠中，AAI處理對小鼠腎臟組織的ACE活性變化趨勢與WT小鼠相似，但上升的chymase活性和表現均較WT小鼠顯著為大。在AAI誘發腎臟病變的實驗動物中，給予chymase抑制劑並沒有顯著影響腎臟組織中ACE及ACE2的活性變化，但病理組織結果顯示，chymase抑制劑雖不能有效地修復因AAI處理所導致腎小管萎縮之病變，但卻可以顯著降低腎臟間質內的纖維化病灶。 我們的實驗結果顯示，在腎臟中ACE2的缺失並不會加速AAI誘發腎臟病變病程，而抑制chymase的表現與活性，能減緩AAI誘發腎臟病變的病程，這些發現皆有助於醫師與研究者們更了解RAS中各項重要酵素在腎臟疾病中扮演的角色，並據此研發新的腎臟疾病標的藥物。

In kidney, angiotensin converting enzyme (ACE) is the primary enzyme which convert angiotensin I (Ang I) to angiotensin II (Ang II) in renin-angiotensin system (RAS). However in some renal diseases, several evidences have showed that chymase that hydrates Ang I to Ang II independent to ACE may play an important role. The aim of this study is to know whether chymase plays a crucial role in aristolochic acid I (AAI)-induced nephropathy. General RAS inhibition, such as ACE inhibitor (ACEI) and Ang II receptor blockers (ARBs) treatment, is effective in reducing renal disease progression in early nephropathy, but they provide limited protection at a later stage. Due to pivotal role of chymase in the pathogenesis of cardiovascular and renal disease, this study tested the effect of chymase inhibition on the disease progression in the mice with aristolochic acid I (AAI)-induced nephropathy. Animal model of nephropathy was performed by using wild type (C57BL/6, WT) and ACE2 knockout (ACE2 KO) mice. The mice were treated with AAI via intraperitoneal injection and the accumulated AAI dosages are 45 and 100 mg/kg of body weight. The animal were sacrificed after another 24 hours or 2 weeks for acute and chronic nephropathy development, respectively, the blood and kidney were harvested for further biochemical, pathologic and molecular assays. Both of WT and ACE2 KO mice after the challenge of AAI, decreased body weight and enhanced serum creatinine as well as increased blood urea nitrogen were observed. In the renal tissue sections, infiltrating inflammatory cells and fibrosis in renal interstitial tissue were observed. Above biochemical and pathologic characterizations in the ACE2 KO mice were more severe than those in the WT mice. Increased MMP-2 and decreased MMP-9 in renal tissue were found in the WT mice treated with AAI compared with those in the control mice. Similar to the biochemical and pathologic changes, the changes on renal gelatinase activities in the ACE2 KO mice were more significant than those in the WT mice. Renal RAS related enzyme activities in the mice with AAI treatment were detected. Chymase activity was significantly increased and ACE activity was significant decreased after AAI treatment. Opposite to ACE, renal ACE2 activity was enhanced by AAI treatment. In the ACE2 KO mice, the ACE activity change is similar with that in the WT mice, but increased chymase activity was more significant compared with that in the WT mice. In the mice with AAI-induced nephropathy, the treatment of chymase inhibitor could reduce the chymase activity induced by AAI challenge, but could not effect on the renal ACE and ACE2 activities. In the histological findings, chymase inhibitor could not reverse the tubules atrophy in renal, but it could reduce the fibrotic lesions in the interstitial renal tissue. The experimental results show that, in the intrarenal environment, ACE2 deficiency would not exacerbate the AAI-induced nephropathy. And AAI-induced nephropathy progression was mitigated with chymase inhibiter treatment. This study may help physicians and researchers to know more information about the role of RAS in renal pathogenesis and can be applied in the drugs development of new targets for nephropathy.