探討第二型血管收縮素轉化酶與凝乳酶在糖尿病腎病變病程之扮演角色

Abstract

糖尿病腎病變(diabetic nephropathy, DN)是慢性腎臟疾病(chronic kidney disease, CKD)的主要病因之一，過去文獻指出腎素-血管收縮素系統(renin angiotensin system, RAS)中 angiotensin II (Ang II)的調節失常，是加速 DN 病程發展的主因之一。據此，本研究目的為探討在糖尿病(diabetes mellitus, DM)誘發之腎病變過程中第二型血管收縮素轉化酶(angiotensin converting enzyme II,ACE2)與凝乳酶(chymase)扮演之角色。 本研究利用 BKS.Cg-Dock7 m +/+ Lepr db /JNarl 小鼠(db/db)與 ACE2 基因剔除小鼠(ACE2 KO)作為實驗模型之動物，並成功地利用這兩種小鼠配種出 db 與 ACE2 雙基因剔除小鼠(db & ACE2 KO)，應用於 DN 誘發模型之研究。我們使用Streptozotocin (STZ)腹腔注射以誘發 ACE2 KO 小鼠的胰島 β 細胞死亡，使實驗小鼠作為第一型糖尿病(Type 1 DM, T1DM)；可自發性誘發高血糖的 db/db 小鼠則作為第二型糖尿病(Type 2 DM, T2DM)之研究。在上述兩種實驗動物模型中，小鼠於 8 周齡持續餵予高脂飼料(high-fat diet, HFD)4 周以加速糖尿病腎病變的病程發展。在 4 周腎病變誘發期後，犧牲 8 周齡及 12 周齡小鼠並收集其血液、尿液、腎臟及胰臟組織，分別進行生化、組織病理及分子檢測。 經 HFD 餵予的 db/db 小鼠，其血清總膽固醇和尿液白蛋白較餵予正常飼料的db/db 小鼠顯著提高，而腎臟組織切片也顯示腎絲球間質擴散的病變情形較為嚴重。而以 STZ 與 HFD 處理誘發 ACE2 KO 小鼠高血糖後，小鼠的血清三酸甘油酯、總膽固醇、尿素氮及尿液白蛋白均顯著上升，腎臟組織中則呈現大量白血球細胞浸潤的炎症反應病徵，而腎絲球間質擴散情形只有些微提升。db & ACE2 KO小鼠在 HFD 誘發 DN 的各項生化指標上，均顯著較 db/db 小鼠嚴重。 在腎臟組織中 RAS 相關酵素之活性變化顯示，餵予 HFD 會造成 db/db 小鼠腎臟組織的 ACE 和 chymase 活性顯著上升，ACE2 活性則維持不變。由 STZ 誘發 T1DM 之 ACE2 KO 小鼠顯示腎臟 ACE 活性提高，但不會影響 chymase 活性，而只餵予 HFD 的小鼠腎臟組織中 ACE 和 chymase 活性均會顯著提高。經 HFD餵予後，db & ACE2 KO 小鼠腎臟組織中的 ACE 活性相較於 db/db 小鼠顯著提高，但 chymase 活性不變。 實驗結果顯示 ACE2 基因剔除與糖尿病腎病變下的 chymase 活性變化有關且會加速 db/db 小鼠的 DN 病程發展，因此本研究所建立的 db & ACE2 KO 小鼠相較於 db/db 小鼠更易於作為 DN 之實驗動物模型。上述的實驗結果有助於醫師與研究者們更了解 RAS 中各項重要酵素在 DN 疾病中扮演的角色，並據此研發新的腎臟疾病標的藥物。

Diabetic nephropathy (DN) is a major cause of chronic kidney disease (CKD). Previous researches indicated that imbalanced angiotensin II (Ang II) in renin angiotensin system (RAS) can accelerate the progression of DN. Therefore, the aim of this study is to explore the roles of angiotensin converting enzyme II (ACE2) and chymase play in the progression of diabetes mellitus (DM)-induced nephropathy. In this study, animal models of DN were performed by using BKS.Cg-Dock7m+/+Leprdb/JNarl (db/db) mice and ACE2 gene knockout (ACE2 KO) mice. Additionally, we successfully generated db & ACE2 double gene KO (db & ACE2 KO) mice by a series of mating and breeding strategy using db/+ mice and ACE2 KO mice. The Type 1 DM (T1DM) of ACE2 KO mice was induced by intraperitoneal injection of Streptozotocin(STZ), a chemical to damage the pancreatic β-cells of mice. The Type 2 DM (T2DM) animal model was performed in db/db mice that high blood glucose level can be spontaneously induced. To accelerate the progression of DN, both of the animal models were subjected to high-fat diet (HFD) feeding at 8-week-old. The mice were sacrificed after 4 weeks chronic nephropathy development, and the blood, urine, kidney and pancreas of 8-week or 12-week-old mice were collected for further biochemical, pathologic and molecular assays. In the group of db/db mice fed with HFD, serum total cholesterol and urine albumin were significantly increased, and severe mesangial matrix expansion was also observed compared with those of the db/db mice fed with normal diet. After high blood glucose in ACE2 KO mice was successfully induced by STZ and HFD treatment, serum triglycerides, total cholesterol, blood urea nitrogen and urine albumin were significantly increased. In these mice, severe inflammation symptoms, inflammatory cells infiltrating in renal interstitial tissue, was observed, but there is slightly increasing in mesangial matrix expansion. Above biochemical and pathologic characters were more severe in db & ACE2 KO mice compared with those found in db/db mice. Renal RAS-related enzyme activities in the db/db mice fed with HFD, show that ACE and chymase activities were significantly increased, but ACE2 activity remained insignificant change. The ACE activity was increased in ACE2 KO mice by STZ treatment, but renal chymase activity was no significant change. After HFD feeding, both of renal ACE and chymase activities in ACE2 KO mice were significantly increased. The renal ACE activitiy was significantly increased but chymase activity was similar in db & ACE2 KO mice that compared with those found in db/db mice. Our results show that ACE2 gene knockout related to the change of chymase activity in DN and accelerate the progression of DN in db/db mice. Therefore, db & ACE2 KO mice is easier to establish an animal model of DN compared with db/db mice. The discoveries of our study can help physicians and researchers to know more information about the role of RAS in renal pathogenesis, and the information can be applied in the new drug development for nephropathy.