Expression Of HO-1and P47-phox In Atherosclerotic Renal Artery Stenosis And Interferenced By Antihypertensive Drugs

Background and objective: Autherosclerosis and fibromuscular dysplasia（FMD） is the most common reasons of renal artery stenosis. Compared withFMD, atherosclerotic renal artery stenosis（ARAS） was easier to damage kidney.ARAS can lead to kidney damage through a variety of ways, studies in recentyears have found that oxidative stress is one of the main pathway. There aremany studies found that heme oxygenase-1（HO-1） which protect kidney alsohas the physiologial effect of anti-oxidation, but less studies also observed thatHO-1can protect kidney in atherosclerotic renal artery stenosis （ARAS）, so it’sworth to be studied. Antihypertensive drugs can lowering blood pressure whichalso have the role of reduce target organ damage, but the effect inatherosclerotic renal artery stenosis is still controversial. So it is worth to bestudied that whether the antihypertensive drugs can reduce kidney damagethrough regulate the expression of HO-1and NADPH oxidase, and intervenethe oxidative stress in kidneys. Therefore, we use operation method to copy ofthe single and renal artery stenosis and combined high fat feeding to establishthe model of atherosclerotic renal artery stenosis. By measuring the change ofHO-1and NADPH oxidase in the left kidney, to explore the protective functionof kidney and its possible mechanism about difference antihypertensive drugs（direct renin inhibitors and calcium channel blockers）, the purpose is to provide a theoretical basis and strategies for the effective treatment of renal arterystenosis.Methods:（1） The rat atherosclerotic renal artery stenosis animal model:36male SD rats were randomly divided into Two Kidney One Clip,2K1C andcontrol group.2K1C group were renal artery stenosis by parallel andacupuncture needles method,the control group just exposed to the left renalartery. All the rats were injected into VitD₃0.5million IU/kg and feeded withhigh fat diet to establish the model of atherosclerotic renal artery stenosis in10weeks.（2）Study the protective function of kidney in the model of atheroscleroticrenal artery stenosis: After10weeks, the rats of control group and2K1C groupwere randomly grouped and given intervention: saline group, aliskiren groupand amlodipine group, after4weeks,following test like these:1)Detectionblood triglyceride, cholesterol, urea nitrogen and serm creatinine by theautomatic biochemical analyzer;2)The kidney were cut, part of it PAS stainingand immunohistochemical; the other part to be cryopreservation at-70℃.3)immunohistochemical detection of HO-1and P47-phox expression:SP methodwas used to detect the change of HO-1and P47-phox’s expression in the kidney.Using Image-pro plus6.0software analysis.4)RT-PCR detect the expression ofHO-1and P47-phox: to extract the total RNA of kidney which was stored at-70℃, reverse transcription, amplification,electrophoresis, then use Quangtity onephoto software to analysis. Results:（1）The method of parallel acupuncture needles combined withfeeded high-fat diet and injected VitD₃50million IU/kg can effectivelyestablished the animal model of atherosclerosis and renal artery stenosis in10weeks.（2）The biochemical analyzer results of serum lipids and renal function:Compared with the previous model, the blood cholesterol（TC） weresignificantly higher after surgery（P<0.05）;compared with the saline group aftersurgery, the serum level of TC increased significantly in aliskiren group aftersurgery （P<0.05）,and the increased TC level was not significantly difference inamlodipine group after surgery（P>0.05）;The TC level was also increasedsignificantly in aliskiren group and amlodipine group aftersurgery（P<0.05）.Compared before and after model,the serum creatinine（Scr）higher levels in the saline group and amlodipine group were statisticallysignificantly （P<0.05）,while aliskiren group before and after difference werenot significantly（P>0.05）;Compared with the saline group after surgery,the Scrlevel increased significantly differences in aliskiren group after surgery（P<0.05）;the difference between aliskiren group and amlodipine group aftersurgery were not significantly（P>0.05）.（3） The changes of HO-1and P47-phox protein expression in experimentalrat’s kidney：1)The expression of HO-1in the kidney：There’s no expression ofHO-1in normal control group.HO-1expressed in the proximal tubule epithelial cells and medullary ascending branch of crude segment epithelial cells of the2K1C group rat’s kidney. There are significantly differences between the salinegroup and aliskiren group or amlodipine group,and the aliskiren group andamlodipine group（P<0.05）.2)The expression of P47-phox: There’s noexpression of P47-phox in the normal control group. It can be seen in thekidney glomerular endothelial cells, mesangial cells and tubular of2K1C group.The result is contrary to HO-1.The expression of P47-phox is the most in thesaline group and less in the aliskiren group. The difference is statisticallysignificantly, P<0.05.（4）The changes of HO-1, P47-phox’s mRNA experimental rat’s kidney: Inthe normal control group, there’s no expression of HO-1and P47-phox. Theexpression of HO-1is increased in aliskiren group and amlodipine group, andthe expression in aliskiren group is more than in the amlodipine group. Theexpression of P47-phox is decreased in aliskiren group and amlodipine group.The results are the same to the immunohistochemical.Conclusion:（1）The method of narrow parallel acupuncture needlecombined with feeded high-fat diet and injected VitD₃can effectively establishthe animal modlel of atherosclerosis and renal artery stenosis.（2） In the ARASkidney,HO-1can be induced to express and inhibit oxidative stress to protectrenal function.（3）Aliskiren and amlodipine can induce the expression of HO-1and inhibit the expression of p47-phox, that can inhibit oxidative stression andreduce the renal damage. Aliskiren may be superior to amlodipine.