尿毒症患者血液中及其經血液透析後血液中MMPs/TIMPs和ACE/ACE2活性比值之變化

Abstract

研究目的： 腎素-血管擴張素系統(renin-angiotensin system; RAS)和基質金屬蛋白酶 (matrix metalloproteinases; MMPs) 與其組織抑制因子 (tissue inhibitor of MMPs; TIMPs) 在心臟與腎臟疾病的病理機轉中扮演重要作用。血管收縮素轉化酶(angiotensin-converting enzyme; ACE)與第二型血管收縮素轉化酶 (angiotensin- converting enzyme II; ACE2)是RAS中重要的胜肽酶，但它們卻扮演著互為拮抗功能的角色。據此，在尿毒症洗腎病患(uremic patients)的血液中不平衡的ACE/ACE2及MMPs/TIMPs比值，可能和患者的血管病變有關。據此，本研究檢測尿毒症患者血漿中ACE、ACE2、MMPs及TIMPs活性或濃度的改變，並探討這些患者在單一次血液透析後，血液中這些生物因子的變化。

實驗方法： 本研究共收集372位尿毒症患者之血液透析前與血液透析後的血液，測量其血液和生化值；另外也收集50位年紀相當健康者的血液樣品，做為各項檢測值的對照。血漿中MMP-2和MMP-9活性以明膠酶譜法(gelatin zymography)檢測之，ACE和ACE2活性使用螢光受質法測定之，TIMP-1、TIMP-2、Ang II及Ang-(1-7)濃度以酶聯免疫吸附試驗法(enzyme-linked immunosorbent assay; ELISA)測定之。

研究結果： 血液檢測結果顯示，與對照組相較，尿毒症患者血漿中有顯著較高的MMP-2和MMP-9活性，以及顯著較低的TIMP-1濃度。因此，在尿毒症患者血漿中有顯著較高的MMP-2/TIMP-2和MMP-9/TIMP-1比值。在單一次血液透析前後的血液檢測結果顯示，血液透析後血漿中MMP-2活性會顯著下降，TIMP-1濃度則顯著升高。雖然血液透析並沒有顯著影響血漿中MMP-9和 TIMP-2濃度，但血液透析後，血漿中MMP-2/TIMP-2和MMP-9/TIMP-1比值均顯著下降。我們也檢測在這群尿毒症患者有心血管疾病病史者(n =122)或無有心血管疾病病史者(n = 250)，其血漿中ACE與ACE2活性，以及血管收縮素II (angiotensin II; Ang II)與血管收縮素1-7 (angiotensin 1-7; Ang-(1-7)的濃度變化。與對照組相較，尿毒症患者血漿中有顯著較高的ACE活性和Ang II濃度，以及顯著較低的ACE2活性和Ang-(1-7)濃度。因此，在尿毒症患者血漿中有顯著較高的ACE/ACE2比值，而這種ACE/ACE2失衡狀況有在有心血管疾病病史的尿毒症患者中更為明顯。在單一次血液透析前後的血液檢測結果顯示，有心血管疾病病史的尿毒症患者之血液透析後血漿中ACE、ACE/ACE2及Ang II呈現上升，而ACE2卻為下降，此結果在無心血管疾病病史的尿毒症患者中卻不顯著。

結 論： 血液透析可以促使尿毒症患者血液中MMP-2活性降低，增加TIMP-1濃度，致使其血液中MMPs/TIMPs比值顯著下降。血液透析卻促使尿毒症患者血液中ACE/ACE2活性比失衡，尤其是在有心血管疾病病史的尿毒症患者，此現象更為顯著。 有心血管疾病病史的尿毒症患者中，血液透析可使患者之ACE/Ang II/AT1R作用途徑上升，使ACE2/Ang-(1-7)/Mas作用途徑下降。此項因血液透析所導致之血液中ACE/ACE2活性比失衡與MMPs/TIMPs改變，均需更多實驗來探討其生理機轉。

Purpose: Renin-angiotensin system (RAS) and matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) play important roles in the pathophysiology of heart and renal diseases. Angiotensin converting enzyme (ACE) and angiotensin converting enzyme II (ACE2) are major peptidases of RAS components and play counteracting functions. Imbalanced ACE/ACE2 and MMPs/TIMPs is implicated in the vascular alterations of uremic patients on hemodialysis (HD). Therefore, we have investigated the levels of plasma ACE, ACE2, MMPs and TIMPs in uremic patients, and the effects in the course of a single HD session on the levels of these factors.

Methods: There were 372 uremic patients on regular HD treatment and 50 healthy controls enrolled in this study. The plasma MMP-2 and MMP-9 activities were detected by gelatin zymography assay, and ACE and ACE2 were assayed detected using fluorogenic substrates assay. Plasma TIMP-1, TIMP-2, Ang II and Ang-(1-7) concentrations were determined by enzyme-linked immunosorbent assay (ELISA).

Results: Significantly higher plasma MMP-2 and MMP-9 and decreasing TIMP-1 in the uremic patients (i.e., HD patients) were detected compared with those in the controls. Therefore, there were markedly higher MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios in the HD patients. In the course of a single HD session, plasma MMP-2 level was significantly decreased from pre-HD to post-HD. TIMP-1 concentration was significantly increased from pre-HD to post-HD. Although the HD session did not have a significant effect on the levels of plasma MMP-9 and TIMP-2, both plasma MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios were significantly decreased from pre-HD to post-HD levels. We also compared pre-HD levels of plasma ACE, ACE2, Ang II and Ang-(1-7) in the HD patients with (n = 122) and without (n= 250) cardiovascular diseases (CVD) to those of the controls (n = 50). The HD patients, particularly those with CVD, showed a significant increase in the levels of plasma ACE and Ang II, whereas plasma ACE2 and Ang-(1-7) levels were lower than those in the healthy controls. Therefore, markedly loss of ACE and ACE2 correlation, i.e., imbalanced ACE/ACE2, was observed in the HD patients with CVD. In the course of a single HD session, the plasma ACE, ACE/ACE2 and Ang II levels in the HD patients with CVD were increased from pre-HD to post-HD. On the contrary, ACE2 level was decreased after the HD session. These changes were not detected in the HD patients without CVD.

Conclusions: HD session could decrease MMP-2 and increase TIMP-1 level in the circulation of uremic patients, that resulted the decreasing MMPs/TIMPs ratio due to HD session. Pathogenically imbalanced circulating ACE/ACE2 was detected in the HD patients, particularly those with CVD. HD session could increase ACE/Ang II/AT1R axis and decrease ACE2/Ang-(1-7)/Mas axis activity in the circulation of HD patients with CVD. The imbalanced ACE/ACE2 and the changes of MMPs/TIMPs ratio due to a single HD session in the HD patients is required to further validate on its mechanisms.