利用動物實驗與健保資料庫分析探討腎素-血管收縮素-醛固酮系統與腎臟病及心血管疾病之關聯性

Abstract

本論文分成兩個部分，第一部分研究主題為末期腎病(end-stage renal disease, ESRD)需要長期透析的病人，其使用血管收縮素受體阻斷劑(angiotensin receptor blockers, ARBs)降低主要不良心血管事件(major adverse cardiovascular events, MACEs)風險效果的探討；第二部分研究主題為利用第一型馬兜鈴酸(aristolochic acid I, AAI)誘導腎臟病變之動物模式探討腎素-血管收縮素-醛固酮系統(renin-angiotensin-aldosterone system, RAAS)中與血管收縮素(angiotensin, Ang)代謝相關之酵素，尤其是糜蛋白酶(chymase)活性的變化。 第一部分研究：MACEs是ESRD需長期血液或腹膜透析病人最常發生的併發症和最主要的死亡原因。許多臨床試驗已經證實ARBs使用於腎功能有異常但還不需要透析的病人上，可以降低發生MACEs的風險，但對於用於長期透析病人的效益與風險並沒有足夠的評估證據。基於以上原因我們設計了這個研究，目的就是要探討是否ARBs用於需長期透析的病人也可以獲得相同的結果。在這個研究中，我們使用全民健康保險資料庫來執行這個全國性配對-世代追蹤，依據資料庫中使用的國際疾病分類第九版中的疾病碼和處置碼來篩選出需長期透析，之前沒有MACEs，且在開始追蹤前的6個月沒有使用過ARBs的病人。 經過篩選，共有1,800位病人被納入此次研究追蹤，其中有1,061位從未使用過ARBs，224位使用1-90天，515位使用超過90天。研究追蹤發現ARBs使用於長期透析病人的確可以顯著的降低了MACEs的發生率，進而也減少了病人需要執行侵入性檢查治療的次數以及其所帶來的術式併發症與健保醫療花費。而其產生效果的時間在不同的共病族群中有所不同：(1) 有高血壓而沒有糖尿病和高血脂的病人，使用超過1,095天以上的ARBs可以顯著降低MACEs；(2) 有高血壓合併糖尿病者，使用超過365天就有效果；(3) 有高血壓合併高血脂者，使用超過365天就有效果，但效果卻在使用超過1,095天後消失；(4) 有高血壓合併糖尿病和高血脂者，只要使用超過90天就有效，而且這效果是持續的。基於以上發現我們建議年紀超過50歲以上，之前未有發生過MACE的高血壓透析病人，尤其是又合併有糖尿病與高血脂疾病時，可以將ARBs當成透析病人降血壓的第一線用藥。基於以上的研究，我們認為可以設計一個隨機對照的臨床試驗去進一步證實在這個研究中的發現。 第二部分研究：在RAAS中，血管收縮素轉換酶(angiotensin converting enzyme, ACE)為將Ang I轉換成Ang II的主要途徑；然而在許多腎臟疾病中，已有證據指出糜蛋白酶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AAI誘導之腎臟病變過程中是否扮演重要的角色。實驗動物方面，我們使用第二型血管收縮素轉換酶(angiotensin converting enzyme II, ACE2)基因剔除(即ACE2-/y)之雄小鼠來進行腎臟病變模型實驗。藉由多次腹腔注射AAI (累積劑量為100 mg/kg/4-weeks)誘發腎臟損傷，再經過2週和4週的病情發展期後，犧牲小鼠收集其尿液、血液和腎臟樣本，進行生化、組織病理及酵素活性檢測。 實驗結果顯示，在AAI誘發腎臟損傷後，可發現小鼠的體重下降，血中的尿素氮(blood urea nitrogen)和肌酸酐(creatinine)上升；在腎臟組織的hematoxylin and eosin (H&E)染色切片中，可發現大量單核球細胞浸潤的炎症反應，而在Masson’s trichrome染色切片中，則發現明顯的腎臟間質纖維化病變。實驗組小鼠腎臟組織的第二型基質金屬蛋白酶MMP-2 (matrix metalloproteinase-2)活性顯著上升，而MMP-9活性則是下降。最重要的，我們發現chymase的活性有顯著上升，但是腎臟ACE的活性卻未因AAI誘發腎臟病變而有顯著變化。由此顯現chymase在AAI誘導的腎臟病變機轉中扮演重要性的角色。藉由此發現，我們下一步可以朝向尋找可以抑制chymase的物質，做為專門針對減緩腎臟病變的新藥研發。

There are two parts of topics performed in this study. In the first part, we studied the effects of angiotensin receptor blockers (ARBs) on decreasing the risk of major adverse cardiovascular events (MACEs) in patients with end-stage renal disease (ESRD) on maintenance dialysis. In the second part, we studied the role of angiotensin-related enzymes, especially the activity of chymase, of renin-angiotensin-aldosterone system (RAAS) in nephropathy induced by aristolochic acid I (AAI). Part I of the study: MACEs are the leading cause of morbidity and mortality in patients with end-stage renal disease (ESRD) on maintenance hemodialysis or peritoneal dialysis. Many studies have proved that ARBs, a class of antihypertensive drugs, can reduce the risk of MACEs in the people with normal kidney function or with impaired kidney function without dialysis yet. There are no clear evidences between benefits and risks on dialysis patients using ARBs, so we conducted a study to clarify whether ARBs therapy could also attenuate this risk in patients with ESRD on maintenance dialysis. We used the National Health Insurance Research Database (NHIRD) for this nationwide matched cohort study. Patients were selected under the International Classification of Diseases, Revision 9 (ICD-9) codes, including disease codes and treatment codes. Patients undergoing maintenance dialysis without a history of MACEs and without use of ARBs within the latest 6 months prior to enrollment were recruited. A total of 1,800 patients was enrolled in this study, 1,061 had never used ARBs, while 224 had used them for 1–90 days, and 515 had used them for more than 90 days. This study showed that ARBs can significantly decrease the incidences of MACEs and decrease the demands of invasive procedures of PTCA and PTA for AMI and PAD respectively. The time needed to bring above benefits depended on cumulative prescription days of ARBs in different subgroups：(1) hypertensive patients without diabetes mellitus and hyperlipidemia, it worked when cumulative prescription days exceeds 1,095 days; (2) patients with diabetes mellitus needed more than 365 days; (3) patients with hyperlipidemia needed more 365 days also, but the benefits vanished after 1,095 days; (4) patient with diabetes mellitus and hyperlipidemia, only above 91 days needed and the benefit sustained. Based on this research that ARBs could significantly reduce the occurrence of MACEs, and decrease the demands of invasive procedures, we strongly suggest that ARBs should be prescribed as the first line of antihypertensive to the hypertensive, without MACEs before, dialysis patients who are older than 50, especially coexisting with diabetes mellitus and hyperlipidemia. Based on this study, we think we should furtherly make a randomized controlled trial to confirm these benefits. Part II of the study: Angiotensin converting enzyme (ACE) is the primary enzyme to convert angiotensin I (Ang I) to angiotensin II (Ang II) in RAAS. However, some studies had showed that chymase may play an important role to catalyze Ang I to Ang II, independent of ACE, in kidney disease progress. The aim of this research was to know whether chymase may play the crucial role in AA-induced nephropathy. We used male ACE2 knockout (ACE2 KO) mice (ACE2-/y) for this study. The mice were treated with AAI via intraperitoneal (i.p.) injection and the accumulated AAI dosage was 100 mg/kg/4weeks. The animals were sacrificed 2 weeks and 4 weeks later for kidney disease development, then urine, blood and kidney tissues were sampled for further assays of biochemical, pathological and enzymatic activities. We found that the AAI-treated mice decreased body weight, and increased serum creatinine and blood urea nitrogen levels. In the renal tissue sections, we found high amount of inflammatory cells by hematoxylin and eosin (H&E) stain, and much fibrosis in the tubulointerstitial tissue by Masson’s trichrome stain. The activities of matrix metalloproteinase-2 (MMP-2) increased but MMP-9 decreased in these AAI-treated mice. Most importantly, we found a significantly increased chymase activity in the kidney tissues of these mice, but the ACE activity did not show significant changes, which indicated the chymase may play the important role in the nephropathy progress. We can conduct a new drug research to find some anti-chymase substances to ameliorate the kidney damages.