【作者】 高宁；

【导师】 徐瑞；

【作者基本信息】 山东大学 ， 内科学（心血管病）（专业学位）， 2021， 博士

【摘要】 研究背景高血压肾病是导致终末期肾病的主要因素之一,早期评估肾损害危险因素,对高血压进行综合防治,从而延缓高血压肾病的进展,对阻止终末期肾病的发生具有重要的临床意义。流行病学研究表明,高同型半胱氨酸血症(hyperhomocysteinemia,HHcy)和低叶酸(folic acid,FA)现象在中国高血压人群中普遍存在,而HHcy可协同增加高血压患者动脉粥样硬化、脑卒中等疾病的发生。但关于HHcy对高血压肾损害方面的研究甚少,我们的前期临床研究已证实HHcy可增加高血压患者的蛋白尿,但HHcy加重高血压肾损害的机制尚不明确。烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶是生理条件下活性氧产生的主要途径,越来越多的研究发现Hcy可通过NADPH氧化酶参与肾损伤的病理生理过程。足细胞损伤被认为是引起肾小球病变和蛋白尿的关键因素,那么在高血压中HHcy能否通过NADPH氧化酶导致足细胞损伤,进而增加蛋白尿有待进一步探索。Nephrin是足细胞表面的重要结构分子,在维持足细胞形态以及肾小球滤过功能方面起着重要作用;另外它也是重要的信号转导分子,其胞内段被磷酸化后可与多种蛋白相互作用,其中与CD2AP结合后,可激活PI3K/Akt信号转导通路,参与足细胞凋亡的调控。高糖、血管紧张素Ⅱ及球内高压等多种因素均可导致足细胞表面nephrin异常表达,但Hcy能否通过氧化应激调控nephrin表达及其信号转导,参与足细胞凋亡,进而恶化高血压肾病目前尚无报道。目前最安全、有效的降低同型半胱氨酸(homocysteine,Hcy)的方法是补充叶酸。中国脑卒中一级预防试验中的肾脏亚组研究表明,高血压患者补充FA可以减缓慢性肾功能损害。然而目前干预Hcy的FA剂量存在争议,不同剂量的FA干预对肾功能的影响及机制尚不清楚。因此,探讨Hcy对高血压肾损害的作用机制,揭示不同剂量FA的疗效及可能机制,将有利于延缓高血压肾损害的发生,并为高血压合并HHcy的患者叶酸的应用提供进一步的理论依据。为明确阐述以上问题,本课题通过以下两个部分进行探讨:第一部分 高同型半胱氨酸血症对高血压大鼠肾脏的影响及机制研究目的通过建立合并高同型半胱氨酸血症的高血压动物模型,研究HHcy对高血压肾损害的影响,并探讨氧化应激参与HHcy所致的肾损伤的分子机制。方法将Wistar Kyoto(WKY)大鼠随机分为正常对照组(WKY组)和HHcy组(WKY+HHcy 组),将自发性高血压大鼠(spontaneously hypertensive rats,SHRs)随机分为高血压组(SHR组)和高血压合并HHcy组(SHR+HHcy组),每组8只。构建高同型半胱氨酸血症模型,给予WKY+HHcy组和SHR+HHcy组的大鼠2%DL-Hcy(5 ml/kg,每日两次)腹腔注射12周。定期监测血压、血浆Hcy水平;检测肾小球滤过率(glomerular filtration rate,GFR)和尿白蛋白肌酐比值(urinary albumin creatinine ratio,UACR)以评估肾功能变化,进行肾脏PAS染色观察肾小球结构损伤;测量氧化应激指标血清超氧化物歧化酶(superoxide dismutase,SOD)和血清丙二醛(malondialdehyde,MDA)浓度,同时利用 qRT-PCR和Western blot法检测肾脏中NOX2和NOX4的表达,以评价肾脏局部氧化应激状态;采用qRT-PCR和免疫组化法分别检测足细胞nephrin的mRNA和蛋白表达。结果1.腹腔注射DL-Hcy的WKY和SHR大鼠,血浆Hcy浓度明显高于各自对照组,证明成功构建了合并HHcy的高血压动物模型。对各组大鼠第0周、第6周和第12周血压进行重复测量方差分析,结果显示随着时间的增加,Hcy对WKY大鼠和SHR大鼠的收缩压和舒张压的影响无统计学差异(P>0.05)。2.HHcy和高血压均可使尿蛋白增加,肾小球滤过率降低,并且高血压合并HHcy组的尿蛋白水平最高,肾小球滤过率最低,说明两个因素在肾功能损害上起协同作用;但高血压和Hcy两个致病因素中,仍以高血压导致的肾损害为主。肾脏PAS染色显示,WKY+HHcy组肾小球肥大,肾小球细胞外基质略增加;SHR组肾小球的毛细血管袢起皱,肾小球球囊间隙增宽;而SHR+HHcy组肾小球结构损伤最重,肾小球的毛细血管袢明显皱褶、增厚,肾小球细胞外基质增加。3.Hcy和高血压均可使血清SOD水平下降,MDA水平增加,而高血压合并HHcy时,SOD水平更低,MDA水平更高。Hcy和高血压均会导致氧化和抗氧化过程的不平衡,导致慢性氧化应激状态。4.qRT-PCR和Western blot法检测肾组织中NOX2和NOX4的mRNA和蛋白表达水平,结果显示Hcy上调肾组织中NOX2和NOX4的表达;高血压亦上调肾组织中NOX2和NOX4的表达;而高血压合并HHcy时肾脏局部氧化应激最强,肾组织中NOX2和NOX4的表达水平分别高于WKY+HHcy组和SHR组,Hcy协同高血压增强了肾脏局部氧化应激。5.qRT-PCR和免疫组织化学结果显示,Hcy下调足细胞表面nephrin的mRNA和蛋白表达;高血压合并HHcy时足细胞表面nephrin的表达最低,分别低于WKY+HHcy组和SHR组,随着肾脏局部氧化应激的增强,nephrin的表达降低。结论1.成功构建合并HHcy的高血压动物模型,发现HHcy可导致早期肾损害,并与高血压在肾损害方面起协同作用;2.HHcy可引起肾脏组织中GAPDH氧化酶亚基NOX2和NOX4过表达以增强氧化应激,且此作用独立于血压之外;3.HHcy通过增强肾脏局部氧化应激,诱导足细胞nephrin表达减少,进而引起蛋白尿,协同高血压加重肾损害。第二部分 不同剂量叶酸对高同型半胱氨酸血症所致的高血压大鼠肾损害的作用及机制研究目的进一步探讨Hcy参与足细胞损伤的机制,并阐明不同剂量叶酸干预对Hcy所致的高血压大鼠肾损害的作用及机制。方法将SHRs随机分为对照组、HHcy组、HHcy+低剂量叶酸组(HHcy+LFA组)、HHcy+高剂量叶酸组(HHcy+HFA组),每组8只。所有的动物给予腹腔内注射药物12周,并于实验第5周开始同时给予灌胃处理,持续8周,具体方法如下:1)对照组:给予生理盐水(physiological saline,PS,5 ml/kg,每日2次)腹腔注12周+PS(0.5 ml/d)灌胃 8 周;2)HHcy 组:给予 2%DL-Hcy(5 ml/kg,每日 2 次)腹腔注射 12 周+PS(0.5 ml/d)灌胃 8 周;3)HHcy+LFA 组:给予 2%DL-Hcy(5 ml/kg,每日2次)腹腔注射12周+FA(0.4 mg/kg/d)灌胃8周;4)HHcy+HFA组:给予2%DL-Hcy(5 ml/kg,每日2次)腹腔注射12周+FA(4 mg/kg/d)灌胃8周。分别于第4周、第8周和第12周监测血压和血浆Hcy水平。第12周取大鼠腔静脉血及尿液进行血浆肌酐、血清MDA、血清SOD、UACR和GFR检测。进行PAS染色观察肾小球损伤情况,并计算肾小球硬化指数,电镜观察足细胞超微结构的变化。采用qRT-PCR和western blot检测肾脏NADPH氧化酶亚单位NOX2和NOX4的表达,采用免疫组化和qRT-PCR法检测肾组织中nephrin、Akt、Bcl-2和Bax的表达,采用TUNEL法检测足细胞凋亡。结果1.在高血压合并HHcy动物模型基础上,给予不同剂量叶酸干预,发现大、小剂量叶酸均可显著降低血浆Hcy水平,但与小剂量叶酸相比,大剂量叶酸在降低血浆Hcy方面未见更好疗效,增加叶酸剂量未出现合理的量效关系。另外,Hcy和叶酸干预后,高血压大鼠的收缩压和舒张压未见明显变化。2.叶酸可显著降低高血压合并HHcy大鼠的蛋白尿,并改善肾小球滤过率;与小剂量相比,大剂量叶酸对尿蛋白和肾小球滤过率的改善未见明显差异,小剂量叶酸即可对Hcy所致的高血压肾损害具有保护作用。3.肾脏PAS染色显示,HHcy协同高血压加重肾小球结构损伤,表现为肾小球毛细血管袢明显皱褶、增厚,肾小球细胞外基质增加,肾小球硬化指数增高;叶酸干预后肾小球细胞外基质减少,肾小球硬化指数降低,但大、小剂量叶酸组间肾小球结构差异不明显。4.叶酸增加高血压合并HHcy大鼠血清SOD水平,降低血清MDA水平,但大、小剂量叶酸的作用未见明显差异,小剂量叶酸即可改善Hcy导致的慢性氧化应激状态。5.qRT-PCR结果显示叶酸抑制Hcy诱导的肾组织中NOX2和NOX4的mRNA表达的上调;Western blot分析显示叶酸抑制Hcy诱导的肾组织中NOX2和NOX4蛋白表达的上调。而且与小剂量叶酸比较,大剂量叶酸更能有效降低NOX2的表达,但Hcy水平在大、小剂量叶酸组间无明显差异,叶酸发挥了独立于Hcy的抗氧化作用。6.免疫组化染色及qRT-PCR结果显示叶酸干预上调了高血压合并HHcy大鼠足细胞表面nephrin的表达,但与小剂量叶酸相比,大剂量叶酸对增加nephrin无明显优势。7.透射电镜观察足细胞超微结构变化,显示高血压组肾小球基底膜增厚,足细胞足突部分融合;而高血压合并HHcy组的大鼠足突刷状结构消失并广泛融合;叶酸干预后随着nephrin表达的恢复,足突超微结构明显改善。8.qRT-PCR结果显示,Hcy降低高血压大鼠肾脏中Akt和抗凋亡因子Bcl-2的mRNA表达,并增加促凋亡因子Bax的mRNA表达水平,Bcl-2/Bax降低;叶酸干预增加了肾组织中Akt和Bcl-2的mRNA表达,而降低了 Bax的mRNA表达,Bcl-2/Bax升高。免疫组化结果也显示HHcy降低了高血压大鼠肾小球中活化的Akt和Bcl-2蛋白的表达,增加了 Bax蛋白的表达,叶酸干预可对抗HHcy诱导的上述变化。但Akt、Bcl-2和Bax的表达在大、小剂量叶酸组间无显著差异。9.TUNEL法分析足细胞凋亡,结果显示Hcy增加高血压大鼠TUNEL 阳性细胞数量,叶酸治疗后足细胞凋亡减少,但大、小剂量叶酸对足细胞凋亡影响未见明显差异。结论1.Hcy通过增强肾脏氧化应激,诱导足细胞nephrin表达减少,继而下调Akt/Bcl-2信号通路,参与足细胞结构破坏与凋亡,协同加重高血压肾损害;2.叶酸通过逆转Hcy诱导的氧化应激,上调足细胞中nephrin/Akt/Bcl-2信号通路,抑制足细胞凋亡;3.小剂量叶酸即可通过降低Hcy水平以及独立的抗氧化作用,改善HHcy相关性高血压肾损害。更多还原

【Abstract】 BackgroundHypertensive nephropathy is one of the main factors leading to end-stage renal disease.Early assessment of the risk factors of renal damage and comprehensive prevention and treatment of hypertension can delay the progression of hypertensive nephropathy and have important clinical significance in preventing the occurrence of end-stage renal disease.Epidemiological studies have shown that hyperhomocysteinemia(HHcy)and low folic acid(FA)are common in Chinese patients with hypertension,and HHcy can synergistically increase the incidence of atherosclerosis,stroke and other diseases in patients with hypertension.However,there are few studies on HHcy’s effect on hypertensive renal damage.Our previous clinical studies have confirmed that HHcy can increase proteinuria in patients with hypertension,but the mechanism of HHcy aggravating renal damage in hypertensive patients remains unclear.Under physiological conditions,NADPH oxidase(NOX)is the main source of ROS production.More and more studies have found that Hcy can participate in the pathophysiological process of kidney injury through NADPH oxidase.Podocyte injury is considered to be a key factor causing proteinuria and glomerular sclerosis.Whether HHcy can cause podocyte damage through NADPH oxidase in hypertension,thereby increasing proteinuria remains to be explored.Nephrine is an important structural molecule on the surface of podocytes and plays an important role in maintaining podocyte morphology and glomerular filtration function.In addition,it is also an important signal transduction molecule.When phosphorylated,its intracellular segment can interact with a variety of proteins.When bound to CD2AP,it can activate PI3K/Akt signal transduction pathway and participate in the regulation of podocyte apoptosis.Various factors,such as high glucose,angiotensin and high pressure in glomerular spheres,can lead to abnormal expression of nephrin on the surface of podocytes.However,it has not been reported whether Hcy can regulate the expression of nephrin and its signal transduction through oxidative stress,participate in podocyte apoptosis,and further worsen hypertensive nephropathy.FA is the safest and effective way to reduce homocysteine(Hcy).In the renal subgroup study of the Chinese Stroke Primary Prevention Trial,FA supplementation in patients with hypertension can alleviate chronic renal function impairment.However,the FA dose for Hcy intervention is controversial at present,and the effect and mechanism of different doses of FA intervention on renal function are still unclear.Therefore,to explore the mechanism of action of Hcy on hypertensive renal damage and reveal the efficacy and possible mechanism of different doses of FA will be conducive to delaying the occurrence of hypertensive renal damage.It also provides further theoretical basis for the application of folic acid in patients with hypertension complicated with HHcy.In order to clarify the above problems,this topic is discussed through the following two parts:PART ONEEffect of hyperhomocysteine on renal function of hypertensive rats and itsmechanismObjectiveBy establishing a hypertensive animal model with HHcy,the effect of Hcy on renal damage in hypertension was studied,and the molecular mechanism of oxidative stress involved in Hcy-induced renal damage was explored.MethodsWistar Kyoto(WKY)rats were randomized into normal control group(WKY group)and hyperhomocysteinemia group(WKY+HHcy group),and spontaneously hypertensive rats(SHRs)were randomly divided into hypertensive group(SHR group)and hypertension combined hyperhomocysteinemia group(SHR+HHcy group),with 8 rats in each group.Rats in WKY+HHcy group and SHR+HHcy group were given 2%DL-Hcy(5 ml/kg,twice daily)intraperitoneal injection for 12 weeks to construct a hyperhomocysteinemia model.Blood pressure and plasma Hcy levels were monitored.Glomerular filtration rate(GFR)and urinary albumin creatinine ratio(UACR)were measured to evaluate renal function changes,and renal PAS staining was performed to observe glomerular structural damage.Serum superoxide dismutase(SOD)and malondialdehyde(MDA)levels were measured,and the expressions of NOX2 and NOX4 in kidney were detected by qRT-PCR and western blot to evaluate the status of local oxidative stress in kidney.The mRNA and protein expression of nephrin in podocytes were detected by qRT-PCR and immunohistochemistry,respectively.Results1.The plasma Hey level of WKY and SHR rats injected intraperitoneally with DL-Hcy was significantly higher than that of the respective control group,which proved that the animal model of hypertension complicated with HHcy was successfully established.Repeated measurement analysis of variance was performed on the blood pressure of rats at week 0,week 6 and week 12.The results showed that with the increase of time,the effect of Hcy on the systolic and diastolic blood pressure of WKY rats and SHR rats was not statistically different(P>0.05).2.Both HHcy and hypertension can increase urine protein and decrease glomerular filtration rate,and the hypertension combined with HHcy group has the highest urine protein level and the lowest glomerular filtration rate,indicating that the two factors have a synergistic effect on renal damage effect.However,among the two pathogenic factors of Hcy and hypertension,hypertension is still the main risk factor for renal damage.PAS staining of the kidney showed hypertrophy of the glomerulus and slight increase of the extracellular matrix of the glomerulus in WKY+HHcy group.The glomerular capillary loops in SHR group were wrinkled and the glomerular balloon gap widened.However,the glomerular structure was most damaged in the SHR+HHcy group,the glomerular capillary loops were significantly wrinkled and thickened,and the glomerular extracellular matrix was increased.3.Both Hcy and hypertension can reduce the level of serum SOD and increase the level of MDA.However,when hypertension is combined with HHcy,the level of SOD is lower and the level of MDA is higher.Both Hcy and hypertension can lead to an imbalance of oxidation and antioxidant processes,leading to a state of chronic oxidative stress.4.The mRNA and protein expression levels of NOX2 and NOX4 in renal tissues were detected by qRT-PCR and western blot,and the results showed that Hcy up-regulated the expression levels of NOX2 and NOX4 in renal tissues,and hypertension also up-regulated the expression of NOX2 and NOX4 in renal tissue.The local oxidative stress in the kidney was strongest in the SHR+HHcy group,and the expression levels of NOX2 and NOX4 in the kidney tissues were higher than those in the WKY+HHcy group and the SHR group,respectively,suggesting that Hcy and hypertension synergistic enhance local renal oxidative stress.5.qRT-PCR and immunohistochemical results showed that Hcy down-regulated the mRNA and protein expression of nephrin on podocyte surface.The expression of nephrin on podocyte surface was the lowest in the SHR+HHcy group,which was lower than that of the SHR group and the WKY+HHcy group,respectively.With the increase of local oxidative stress in the kidney,the expression of nephrin decreased.Conclusions1.An animal model of hypertension combined with hyperhomocysteinemia was successfully constructed,and it was found that Hcy can cause early renal damage and play a synergistic role with hypertension in renal damage.2.Hcy overexpresses the GAPDH oxidases subunits NOX2 and NOX4 in renal tissues to enhance oxidative stress,and this effect is independent of blood pressure.3.Hcy can induce the decrease of nephrin expression in podocytes by enhancing local oxidative stress in the kidney,and it can cooperate with hypertension to aggravate kidney damage.PART TWOEffect and mechanism of different doses of folic acid on renal damage induced by hyperhomocysteinemia in hypertensive ratsObjectiveFurther explore the mechanism of Hcy involved in podocyte injury,and clarify the effect and mechanism of different doses of folic acid intervention on Hcy-induced renal damage in hypertensive rats.MethodsThe SHRs were randomized into a control group,HHcy group,HHcy+low dose folic acid group(HHcy+LFA group)and HHcy+high dose folic acid group(HHcy+HFA group),with eight animals per group.All animals were given intraperitoneal injections of drugs for 12 weeks,and were given gavage treatment at the same time from the 5th week of the experiment for 8 weeks.The specific method is as follows:1)control group:physiological saline(PS,5 ml/kg,twice a day)was intraperitoneally injected for 12 weeks and PS(0.5 ml/d)was gavage for 8 weeks;2)HHcy group:2%DL-Hcy(5 ml/kg,twice a day)was intraperitoneally injected for 12 weeks and PS(0.5 ml/d)was gavage for 8 weeks;3)HHcy+LFA group:2%DL-Hcy(5 ml/kg,twice a day)was intraperitoneally injected for 12 weeks and FA(0.4 mg/kg/d)was gavage for 8 weeks;and 4)HHcy+HFA group:2%DL-Hcy(5 ml/kg,twice a day)was intraperitoneally injected for 12 weeks and FA(4 mg/kg/d)was gavage for 8 weeks.Blood pressure and plasma Hcy level were measured at week 4,8 and 12,respectively.At the 12th week,vena cava blood and urine of rats were collected for plasma creatinine,serum MDA,SOD,UACR and GFR detection.PAS staining was performed on the renal tissue sections to observe the glomerular damage and calculate the glomerulosclerosis index(GSI).The ultrastructure of podocytes was observed by electron microscopy.Expression of NADPH oxidase subunits NOX2 and NOX4 in the kidney was detected by qRT-PCR and western blot.Immunohistochemical staining and qRT-PCR were used to detect the expression of nephrin,Akt,Bcl-2 and Bax in renal tissues.The apoptosis of podocytes was detected by TUNEL assay.Results1.On the basis of the animal model of hypertension combined with HHcy,different doses of folic acid were given intervention,and it was found that both high and low doses of folic acid can effectively reduce the plasma Hcy level.However,compared with low-dose FA,high-dose FA did not show a better effect in reducing plasma Hcy,and increasing the dose of folic acid did not show a reasonable dose-effect relationship.In addition,after the intervention of Hcy and folic acid,the systolic and diastolic blood pressure of hypertensive rats did not change significantly.2.Folic acid can significantly reduce albuminuria and improve glomerular filtration rate in hypertensive rats complicated with HHcy.There was no significant difference in the improvement of urinary protein and glomerular filtration rate in high dose folic acid compared with low dose folic acid.Even low dose folic acid can protect Hcy-induced renal damage in hypertensive rats.3.Kidney PAS staining showed that HHcy cooperated with hypertension to aggravate glomerular structural damage,manifested as glomerular capillary loops were significantly wrinkled and thickened,glomerular extracellular matrix increased,and GSI increased.After folic acid intervention,the glomerular extracellular matrix and the GSI decreased,but there was no significant improvement in glomerular structure in the high-dose folic acid group compared with the low-dose group..4.Folic acid increased serum SOD level and decreased serum MDA level in hypertensive rats complicated with HHcy,but there was no significant difference in the effect of high and low doses of folic acid.Low doses of folic acid can improve the chronic oxidative stress caused by Hcy.5.The qRT-PCR results showed that folic acid inhibited the upregulation of Hcy-induced NOX2 and NOX4 mRNA expression in renal tissue.Western blot analysis showed that folic acid inhibited the upregulation of Hcy-induced NOX2 and NOX4 protein expression in renal tissue.Moreover,compared with the low-dose folic acid,the high-dose folic acid was more effective in reducing the expression of NOX2,but Hcy levels were not lower in the high-dose folic acid group than in the low-dose group,indicating that folic acid exerted an antioxidant effect independent of Hcy.6.Immunohistochemical staining and qRT-PCR analysis showed that folic acid intervention upregulated the expression of nephrin on the podocyte surface of hypertensive rats complicated with HHcy.However,compared with the low dose folic acid,the high dose folic acid had no obvious advantage in increasing nephrin.7.The ultrastructural changes of podocytes were observed by transmission electron microscopy.The glomerular basement membrane of the control group(hypertensive)was thickened,and partial fusion of podocyte foot processes was observed.However,the brush-like structure of podocyte foot process disappeared and was widely fused in hypertensive rats complicated with HHcy.After the intervention of folic acid,with the recovery of nephrin expression,the ultrastructure of the foot process was significantly improved.8.The qRT-PCR results showed that Hcy down-regulated the mRNA expression levels of Akt and anti-apoptotic factor Bcl-2,and up-regulated the mRNA expression level of pro-apoptotic factor Bax,and the ratio of Bcl-2/Bax decreased.After folic acid intervention,the mRNA expression of Akt and Bcl-2 in renal tissue increased,while the mRNA expression of Bax decreased,and the ratio of Bcl-2/Bax increased.Immunohistochemical results also showed that HHcy reduced the expression of activated Akt and Bcl-2 proteins and decreased the expression of Bax protein in the glomerulus of hypertensive rats.The intervention of folic acid can counteract the above-mentioned changes induced by HHcy.However,there was no significant difference in the expression of Akt,Bcl-2 and Bax between the high and low dose folic acid groups.9.TUNEL assay was used to analyze the apoptosis of podocytes.The results showed that Hcy increased the number of TUNEL positive cells in hypertensive rats,and the apoptosis of podocytes decreased after treatment with folic acid.However,there was no significant difference between high dose and low dose of folic acid on podocyte apoptosis.Conclusions1.Hcy induces the reduction of nephrin expression in podocytes by enhancing renal oxidative stress,then down-regulates the Akt/Bcl-2 signaling pathway,participates in the structural destruction and apoptosis of podocytes,and synergically aggravates renal damage in hypertension.2.Folic acid inhibited apoptosis of podocytes by reversing oxidative stress induced by Hcy and up-regulating nephrin/Akt/Bcl-2 signaling pathway in podocytes.3.Even low dose of folic acid can improve HHcy-related hypertensive renal damage by reducing Hcy levels and independent antioxidant effects.更多还原