| Background and Objective:Atherosclerosis (AS) is a systemic and progressive intimal disease, the formation and rupture of AS plaque is the main pathology basis with renal artery stenosis (RAS).Currently, the best treatment measures for RAS was still controversial, although use of vascular stenting in clinical can significantly improve blood pressure in the short term, it can protect the target organs of kidney are unclearly. Recent studies have found that some antihypertensive drugs such as calcium channel blockers can lowering blood pressure, it also has the role of anti-AS. As a novel antihypertensive drug,the direct renin inhibitors can blocking renin production from the source, it also has the dual role of lowering blood pressure and anti-AS. However, the sufficient evidence of comparativly study with direct renin inhibitors and calcium channel blockers in the treatment of RAS was deficiency, due to different mechanisms of hypertension about single or bilateral renal artery stenosis,it was difficult to select the appropriate drug treatment of RAS in clinical appears in clinical. Therefore, we use different Operation method to copy of the single and bilateral renal artery stenosis and combined high fat feeding with injecting VitD3 to establish the animal model of atherosclerosis and renal artery stenosis. By measuring the change of blood lipids, renal function and plasma renin activity (PRA), observation and measurement of the changes of the macrophage specific marker antigen CD68, vascular smooth muscle cell phenotype markers a-SMA, and matrix metalloproteinase (MMP) 1,2,9 in aortic plaque, to explore the influence and its possiblly mechanism about the direct renin inhibitors and calcium channel blockers on plaque stability,the propose is to provide a theoretical basis and strategies for select the rational and effective treatment of atherosclerosis, especially the renal artery stenosis in clinical.Method:1,Animal model:78 male SD rats were randomly divided into one kidney, one clip (1K1C) groups, two kidney one clip (2K1C) and control group,1K1C and 2K1C group were renal artery stenosis by parallel and acupuncture needles method, and only exposed the left renal artery in control group. The rats were injected VitD3 0.50 million IU/kg and high fat diet (10% lard+5% sugar +2% cholesterol and 0.5% cholic acid) to establish the model of renal artery stenosis and AS in 10 weeks.2,Study the influence about artery plaque stability of direct renin inhibitors and calcium channel blockers on rat which had renal artery stenosis:After 10 weeks of feeding, the control group and the 1K1C group,2K1C rats were randomly grouped and given interventions:saline group (0.9% NS2ml/d), aliskiren group (50mg/kg.d) and amlodipine besylate group (6mg/kg.d), intervention time is 4 weeks. Following test after 4 weeks treatment in rats,:(1), Detection blood triglyceride, cholesterol, urea nitrogen and serum creatinine by the automatic biochemical analyzer, measured plasma renin activity (PRA) by radioimmunoassay; (2), The aorta and kidney were cut, part of it to be HE staining (vascular tissue) and PAS staining (kidney); part of it to be cryopreservation of-70℃; (3) immunohistochemical detection of CD68,α-SMA and MMP1,2,9 expression: SABC method was used to detect macrophage CD68, a-SMA and MMP1,2,9 in the expression on the plaque,and PBS instead of primary antibody as negative control, plaque area stained brown particles appeared positive. Using Image-pro plus 6.0 software analysis the Images which were collected, comparing each group's average optical density values (Mean optical density, MOD); (4) Western-blot detection of the expression of MMP9:Take the cryopreservation of aortic tissue was extracted in each group protein, denaturation, electrophoresis, transfer of a membrane, incubated with antibody,then dropping light liquid, Quangtity one photo software analysis of gray values about the target band and the internal reference band after imaging, comparative the changes of the expression of MMP9 in artery plaque of rats. Results:1. The method of parallel acupuncture needles combined high-fat diet (10% lard+5%+2% sugar, cholesterol 0.5% cholic acid) with injected VitD3 50 million IU/kg can effectively established the animal model of atherosclerosis and renal artery stenosis in 10 weeks.2. The biochemical analyzer results of Serum lipids and renal function: Compared with the previous modeling, the blood cholesterol (TC) were significantly higher after surgery (P<0.05); compared with the saline group after surgery, The serum level of TC increased significantly different in aliskiren group after surgery (P<0.05), and the increased TC level was not significantly difference in amlodipine group after surgery (P> 0.05); The TC level was also increased significantly in aliskiren group and amlodipine group after surgery (P<0.05). Compared before and after modeling, the serum creatinine (Scr) higher levels in the saline group and amlodipine group were statistically significant (P<0.05), while aliskiren group before and after differences were not significant (P>0.05); compared with the saline group after surgery, the Scr level increased significantly differences in aliskiren group after surgery (P<0.05); the difference between aliskiren group and amlodipine group after surgery were not significant (P> 0.05).3. PRA results were measured by radioimmunoassay:Comparison with the saline group which had the corresponding modeling approach,the level of PRA were significantly decreased in aliskiren group (P<0.05), while the 2K1C and 1K1C amlodipine group was significantly increased (P<0.05); also, the differences between aliskiren group and amlodipine group was significant (P <0.05).4. The results of immunohistochemistry:Compared the average optical density (MOD)of the expression of a-SMA, macrophage CD68 and MMPs in plaque area which had the same mode of operation.①The expression of a-SMA in plaque area:There were significant differences between the saline group and aliskiren group or amlodipine group,and the aliskiren group and amlodipine group. (P<0.05). ②The expression of macrophages CD68in plaque area:2K1C amlodipine group compared with the saline group showed no significant (P> 0.05);but other groups were significant (P<0.05).③The expression of MMPs in plaque area:compared with the saline group,the expression of MMP1,2 in control amlodipine group and 1K1C amlodipine group showed no significant (P> 0.05); Similarly, the expression of MMP9 in 1K1C and 2K1C amlodipine group were no significant (P> 0.05). Other groups were significantly differences (P<0.05).5. Detection of MMP9 expression in the same modeling approach by Western-blot:Compared the target-band gray value/the gray value of internal reference band with the saline group, the expression of MMP9 were significantly decreased in aliskiren group (P< 0.05), while the 1K1C and 2K1C amlodipine group was not statistically significant (P> 0.05), and there were corresponding with immunohistochemical results.Conclusion:1. The method of narrow parallel acupuncture needle combined high fat diet with injection VitD3 can effectively establish the animal models of atherosclerosis and renal artery stenosis.2. The antihypertensive effective were similarly about the direct renin inhibitor aliskiren and the calcium channel blocker amlodipine besylate, but the aliskiren significantly reduced the level of PRA in renal artery stenosis of rat, and it is more useful of inhibition of plasma cholesterol levels.3. Calcium channel blocker amlodipine besylate increase the expression of a-SMA in plaque area, and inhibition of the expression of MMPl, MMP2 in 2K1C group and the expression of macrophage GD68 and MMP9 in control group in plaque area, it is no significant inhibition of the expression of macrophages CD68 and MMP9 in plaque area in 1K1C and 2K1C group, and direct renin inhibitors-aliskiren may also increase the expression of a-SMA and inhibition of the expression of macrophages CD68, MMP1, MMP2, MMP9 in plaque area, suggesting that aliskiren may be superior to amlodipine besylate in plaque stability.
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