GRK4对肾脏脂联素受体促尿钠排泄的调控在高血压中的作用研究

发布时间：2018-04-28 09:47

本文选题：脂联素 + 脂联素受体　；参考：《第三军医大学》2016年博士论文

【摘要】：研究背景目前研究认为,高血压的发病机制涉及多个系统。脂肪组织作为重要的内分泌器官,所分泌的脂肪细胞因子如脂联素、瘦素以及生物活性物质如脂肪酸、前列腺素、血管紧张素原等,在心血管疾病的发生发展中起到了重要作用。脂肪细胞因子的表达以及功能失调,被认为是导致高血压发病的重要原因。在众多的脂肪细胞因子中,脂联素的作用尤其引人注目。脂联素与高血压的关系不仅得到临床结果的佐证,也得到实验动物结果的支持。临床研究发现血浆脂联素水平的改变与高血压密切相关。脂联素敲除小鼠在高盐诱导下表现为血压升高,利用腺病毒过表达脂联素可以降低肥胖小鼠的血压。尿钠重吸收增加,排泄受损是高血压发病中的重要部分,但是脂联素的肾脏作用、参与高血压发生的机制等尚不清楚。脂联素受体分为两个亚型:脂联素受体1(Adipo R1)以及脂联素受体2(Adipo R2)。Adipo R1和Adipo R2在全身广泛分布。研究显示脂联素的两个受体亚型在肾脏均有高表达,并且脂联素通过肾脏脂联素受体对肾脏的生理和病理调节起到了重要作用。我们初期研究也发现脂联素受体在RPT细胞中的表达;肾脏灌注脂联素发挥促尿钠排泄作用,但高血压状态下该作用受损。脂联素受体虽不属于G蛋白偶联受体(G protein coupled receptors,GPCRs),但是却能被G蛋白偶联受体激酶(G protein coupled receptors kinases,GRKs)所磷酸化,其受体磷酸化是其功能改变的重要机制之一;既往的研究发现,在众多的GRK亚型中,GRK4活性增高早于高血压发生而出现,其在高血压中的作用受到人们关注。GRK4基因位于人类染色体4q16.3的位置,与原发性高血压相关。GRK4变异体出现的机率与血压增高程度明显相关。利用GRK4变异体对非洲加纳人原发性高血压的预测准确率达70%以上,而对日本人原发性高血压的预测准确率达94%。我们对多个研究中心的研究结果所做的Meta分析也显示GRK4变异体与高血压发生的关系密切。GRK4变异体三个转基因小鼠均表现为高血压,GRK4?142V小鼠为显性高血压,A486和R65L小鼠在高盐负荷的情况下表现为盐敏感性高血压。以此我们推测GRK4有可能参与了肾脏脂联素受体的磷酸化,从而参与了高血压的进展。所以,针对性抑制肾脏GRK4的表达可作为增强尿钠排泄,从而改善高血压的有效尝试。综上所述,我们推测:脂联素具有利尿排钠作用,该作用在高血压状态下丧失;其丧失的原因归因于脂联素受体发生磷酸化导致受体失活,归因于肾脏GRK4表达和活性增高;抑制肾脏GRK4具有降血压作用。研究目的研究脂联素对肾脏利尿排钠的影响及在高血压发生发展中的作用。明确GRK4是否参与调控脂联素受体磷酸化从而对血压进行调节,超声微泡靶向沉默肾脏GRK4是否对SHR大鼠的血压和尿钠产生影响。研究内容和方法1.脂联素在肾脏尿钠排泄中的作用及其机制1.1采用免疫印迹以及免疫荧光染色的方法,观察脂联素2种受体Adipo R1和Adipo R2,在正常血压大鼠(Wista Kyoto rat,WKY)肾脏及RPT细胞上的表达。1.2利用WKY大鼠,通过单肾灌注不同浓度的脂联素,观察尿流量、尿钠排泄率、以及血压的变化以明确脂联素是否具有利尿作用。同时采用高温灭活脂联素蛋白活性的方法,以排除蛋白的渗透性利尿作用以及致敏作用。1.3在WKY的RPT细胞上,采用Na+-K+-ATP酶活性检测方法,观察脂联素作用不同浓度和时间后对Na+-K+-ATP酶活性的影响。1.4实验首先验证Adipo R1和Adipo R2的小干扰RNA(si RNA)的干扰效率,然后将Adipo R1和Adipo R2的si RNA转染至RPT细胞中,观察在脂联素受体受到抑制时,脂联素对Na+-K+-ATP酶的作用,以明确何种受体介导脂联素的作用。1.5采用Na+-K+-ATP酶活性测定技术,加入磷酸化腺苷酸活化蛋白激酶(Adenosine 5‘-monophosphate-activated protein kinase,AMPK)抑制剂Compound C、一氧化氮合酶(Endothelial nitric oxide synthase,e NOS)抑制剂(NG-nitro-l-arginine methyl ester,L-NAME)初步筛选脂联素抑制Na+-K+-ATP酶活性的信号通路。然后,采用免疫印迹法进一步观察在脂联素作用后,磷酸化AMPK及其下游信号分子e NOS的磷酸化表达情况。2.脂联素受体表达下调及GRK4调控脂联素受体磷酸化导致脂联素促尿钠排泄作用受损2.1采用同WKY大鼠相同浓度梯度的脂联素,对SHR进行单肾灌注。观察尿流量、尿钠排泄率以及平均动脉压的改变。2.2采用自发性高血压大鼠(Spontaneous hypertensive rat,SHR)的RPT细胞,观察脂联素对Na+-K+-ATP酶的作用。2.3采用实时定量PCR观察,比较于WKY RPT细胞,脂联素两个受体基因在SHR RPT细胞上的表达情况,同时,免疫印迹方法检测脂联素受体的蛋白表达情况。2.4单肾灌注脂联素受体激动剂即小分子化合物Adipo Ron对SHR尿钠排泄影响,以进一步明确受损的脂联素利尿作用来源于脂联素受体的改变。2.5根据实验结果,设计脂联素两个受体的过表达质粒,提取质粒并在SHR的RPT细胞上转染,观察脂联素受体恢复表达后,脂联素对Na+-K+-ATP酶的作用。2.6利用免疫共沉淀的方法,观察在SHR的RPT细胞上,脂联素受体的磷酸化表达情况。2.7脂联素受体激动剂Adipo Ron灌注GRK4?142V转基因小鼠(相比较于GRK4?WT小鼠,该小鼠的GRK4活性增强),观察脂联素受体参与的利尿作用是否受损。2.8筛选有效的GRK4 si RNA。观察在SHR RPT细胞中,干扰GRK4表达后,脂联素受体磷酸化程度。3.GRK4超声微泡靶向治疗改善SHR脂联素受体磷酸化并调节尿钠排泄与血压3.1利用5-羧基荧光素(5-Carboxyfluorescein,FAM)标记的超声微泡对GRK4si RNA进行包裹。3.2将超声微泡注射到SHR,在肾脏进行超声微泡靶向破坏技术(Ultrasound-targeted microbubble destruction,UTMD)释放出GRK4 si RNA。20天后,观察SHR血压以及24小时(hour,h)尿量、尿钠排泄率,以及肾功能指标和肾纤维化指标。3.3取UTMD传输GRK4 si RNA至SHR大鼠肾脏,采用免疫共沉淀方法,观察脂联素受体磷酸化情况。研究结果1.脂联素受体Adipo R1以及Adipo R2在肾脏近曲小管均有表达,特别是肾脏皮质,在RPT细胞表达丰富。Adipo R1的蛋白条带为43Kd,Adipo R2的蛋白条带为44Kd。2.脂联素对WKY大鼠具有促尿量和尿钠排泄的作用,该作用呈现浓度依赖性3.脂联素对WKY RPT细胞上的Na+-K+-ATP酶活性产生抑制作用,该作用呈现浓度和依赖性。4.为研究脂联素作用的特异性,采用si RNA在WKY RPT细胞中敲低Adipo R1和Adipo R2,结果发现脂联素对Na+-K+-ATP酶活性的抑制作用消失。5.Na+-K+-ATP酶活性检测发现,加入AMPK阻断剂和e NOS阻断剂后,脂联素对Na+-K+-ATP酶活性的抑制作用消失。同时,脂联素增加了磷酸化AMPK以及e NOS的表达,同时采用AMPK阻断剂后,脂联素对e NOS磷酸化增强的作用消失,说明脂联素通过AMPK-e NOS信号途径对WKY RPT细胞上的Na+-K+-ATP酶活性产生抑制作用。6.脂联素的利尿作用在SHR中受损。脂联素对Na+-K+-ATP酶的抑制作用在SHR的RPT细胞中同样受损。脂联素的作用得到了脂联素受体激动剂Adipo Ron的佐证:即:Adipo Ron具有利尿作用,该作用在SHR丧失。7.SHR肾脏脂联素受体表达量低于对照WKY大鼠,但其受体的表达量改变难以解释其功能丧失,因为过表达Adipo R1和Adipo R2后,其利尿作用仍不能恢复,说明还有其它因素参与。由于脂联素受体的磷酸化影响受体功能,我们的研究发现SHR肾脏脂联素受体磷酸化水平升高,提示高磷酸化水平可能是脂联素受体功能下降的原因。8.为验证GRK4是否参与调控脂联素受体磷酸化过程,我们采用GRK4?142V转基因小鼠灌注脂联素受体激动剂,发现脂联素受体参与调节的利尿作用消失。在SHR的RPT细胞中采用GRK4 si RNA干扰后,脂联素受体Adipo R1和Adipo R2的磷酸化程度减弱。这说明GRK4参与调控了脂联素受体的磷酸化,而脂联素促尿钠排泄作用的受损和脂联素受体在SHR中发生磷酸化相关。9.采用微泡包裹GRK4 si RNA,在SHR的肾脏进行靶向定位超声破碎微泡,释放出的GRK4 si RNA对GRK4产生抑制作用,肾脏脂联素受体的磷酸化水平减弱,SHR大鼠尿钠排泄增加,血压下降。同时,SHR肾功能未受到影响,肾纤维化得到改善。结论脂联素具有促肾脏尿钠排泄作用,在高血压状态下,这一功能受损;检测发现脂联素受体表达下调,但过表达受体后并不能完全恢复脂联素功能。受体的磷酸化可能在其中发挥重要的作用;GRK4活性增高是脂联素受体高磷酸化的原因;UTMD传输GRK4 si RNA有效抑制肾脏GRK4表达,在SHR可发挥降低血压、利尿排钠作用。
[Abstract]:Research background current research suggests that the pathogenesis of hypertension involves multiple systems. Adipose tissue is an important endocrine organ, and the secreted adipocytokines such as adiponectin, leptin and bioactive substances such as fatty acids, prostaglandins, angiotensinogen and so on, play an important role in the development of cardiovascular diseases. The expression and dysfunction of adipocytokines are considered to be an important cause of hypertension. In many adipocytokines, the role of adiponectin is especially attractive. The relationship between adiponectin and hypertension is not only supported by clinical results, but also supported by experimental animal results. Plasma adiponectin is found in clinical research. Level changes are closely related to hypertension. Adiponectin knockout mice are elevated in high salt induced blood pressure. Using adenovirus to express adiponectin can reduce blood pressure in obese mice. The increase in urine sodium reabsorption and impaired excretion is an important part of hypertension, but the renal function of lipoprotein is involved in the pathogenesis of hypertension. The adiponectin receptor is divided into two subtypes: the adiponectin receptor 1 (Adipo R1) and the adiponectin receptor 2 (Adipo R2).Adipo R1 and Adipo R2 are widely distributed throughout the body. The study shows that the two receptor subtypes of adiponectin are highly expressed in the kidney, and the adiponectin is regulated by the renal adiponectin receptor for the kidney's physiology and pathology. Our initial study also found the expression of adiponectin receptors in RPT cells; renal perfusion adiponectin played a role in promoting urinary sodium excretion, but the effect was impaired in hypertension. Although the adiponectin receptor was not a G protein coupled receptor (G protein coupled receptors, GPCRs), it could be found to be G protein coupled receptor kinase (G PR). The phosphorylation of otein coupled receptors kinases, GRKs) is one of the important mechanisms of its receptor phosphorylation. Previous studies have found that in many GRK subtypes, the increase of GRK4 activity is earlier than the occurrence of hypertension, and its role in hypertension is concerned with the location of the.GRK4 gene located in the human chromosome 4q16.3. The incidence of primary hypertension related.GRK4 variants was significantly associated with increased blood pressure. The predictive accuracy of GRK4 variants for essential hypertension in African Garner people was more than 70%, while the accuracy of the Japanese primary hypertension was up to 94%. and the Meta analysis of the results of multiple research centers was also shown by 94%.. The relationship of GRK4 variant with hypertension is closely related to the occurrence of hypertension in three transgenic mice with.GRK4 variant. GRK4? 142V mice are dominant hypertension, and A486 and R65L mice are salt sensitive hypertension under high salt load conditions. We speculate that GRK4 may be involved in the phosphorylation of renal adiponectin receptors. It is associated with the progress of hypertension. Therefore, the targeted inhibition of the expression of GRK4 in the kidney can be used as an effective attempt to enhance urinary sodium excretion and improve hypertension. In summary, we speculate that adiponectin has diuretic excretion of sodium, which is lost in high blood pressure, and its loss of origin is attributed to the phosphorylation of adiponectin receptors to receptors. Inactivation is attributed to the increase of GRK4 expression and activity in the kidney, and the inhibition of renal GRK4 has the effect of lowering blood pressure. The purpose of this study is to investigate the effect of adiponectin on renal diuretic sodium and its role in the development of hypertension. Whether GRK4 participates in the regulation of the phosphorylation of adiponectin receptors and regulates blood pressure, and the ultrasound microbubble target is silent on the kidney GRK4 Influence of blood pressure and urine sodium in SHR rats. Research contents and methods 1. the role of adiponectin in renal urinary sodium excretion and its mechanism 1.1 use immunoblotting and immunofluorescence staining to observe the expression of 2 kinds of adiponectin receptor Adipo R1 and Adipo R2 in normal blood pressure rats (Wista Kyoto rat, WKY) and RPT cells. 1.2 WKY rats were used to infuse different concentrations of adiponectin through single kidney to observe the urine flow, urine sodium excretion rate, and blood pressure changes to determine whether adiponectin has diuretic effect. At the same time, the method of high temperature inactivation of adiponectin protein activity was used to remove the osmotic urination effect and the sensitization of.1.3 on the WKY RPT cells. The effect of Na+-K+-ATP enzyme activity detection method was used to observe the effect of adiponectin on the activity of Na+-K+-ATP enzyme after different concentration and time..1.4 experiment first verified the interference efficiency of small interfering RNA (Si RNA) of Adipo R1 and Adipo R2, and then transfected Adipo R1 and Adipo into the cells, and observed the adiponectin receptor when the adiponectin receptor was inhibited. The role of Na+-K+-ATP enzyme to identify the receptor mediates the action of adiponectin.1.5 using Na+-K+-ATP enzyme activity determination technique, adding phosphorylated adenylate activated protein kinase (Adenosine 5 '-monophosphate-activated protein kinase, AMPK) inhibitor Compound C, one oxygenated nitrogenase (Endothelial nitric) inhibition NG-nitro-l-arginine methyl ester (L-NAME) preliminarily screened the signal pathway of adiponectin inhibiting the activity of Na+-K+-ATP enzyme. Then, the phosphorylation of phosphorylated AMPK and its downstream signal molecule e NOS after the action of adiponectin, the expression of.2. adiponectin receptor down regulation and GRK4 regulation of the adiponectin receptor after the action of adiponectin were further observed. Phosphorylation was damaged by adiponectin induced urinary sodium excretion. 2.1 the same concentration gradient of adiponectin was used in the same WKY rats. Single kidney perfusion was performed on SHR. The urine flow, urinary sodium excretion rate and the change of mean arterial pressure were observed in.2.2 of spontaneously hypertensive rats (Spontaneous hypertensive rat, SHR), and the adiponectin to Na+-K+-ATP was observed. The effect of enzyme.2.3 was observed by real-time quantitative PCR, compared with WKY RPT cells and the expression of adiponectin two receptor genes on SHR RPT cells, and the immunoblotting method was used to detect the protein expression of adiponectin receptor,.2.4 single kidney perfusion adiponectin receptor agonist, that is, the effect of Adipo Ron on SHR urine sodium excretion. One step is to identify the impaired adiponectin diuretic effect from the changes in the adiponectin receptor.2.5. According to the experimental results, the overexpressed plasmids of the two adiponectin receptors were designed, the plasmids were extracted and transfected on the SHR RPT cells. The effect of adiponectin on the Na+-K+-ATP enzyme was observed and the effect of adiponectin on the Na+-K+-ATP enzyme was observed by the method of immunoprecipitation. On the RPT cells of SHR, the phosphorylation of adiponectin receptor,.2.7 adiponectin receptor agonist Adipo Ron perfusion GRK4? 142V transgenic mice (compared with GRK4? WT mice, the GRK4 activity of this mouse is enhanced). After disturbing the expression of GRK4, the phosphorylation of adiponectin receptor.3.GRK4 ultrasound microbubble targeting therapy improves the phosphorylation of SHR adiponectin receptors and regulates the urinary sodium excretion and blood pressure 3.1 using 5- carboxyl fluorescein (5-Carboxyfluorescein, FAM) labeled ultrasound microbubbles for GRK4si RNA to be encapsulated.3.2 into SHR and ultrasound microbubbles in the kidney Ultrasound-targeted microbubble destruction (UTMD) released GRK4 Si RNA.20 after GRK4 Si RNA.20 days, and observed the blood pressure of SHR and the urine volume of 24 hours (hour, H), urine sodium excretion rate, renal function index and renal fibrosis index.3.3 UTMD transfer to kidney, using immunoprecipitation method to observe the adiponectin receptor phosphorus. Results 1. the expression of adiponectin receptor Adipo R1 and Adipo R2 was expressed in the renal proximal tubule, especially in the renal cortex. The protein strip of.Adipo R1 expressed in RPT cells was 43Kd, and the protein strip of Adipo R2 was the effect of 44Kd.2. adiponectin on urinary excretion and urine volume of WKY rats. This effect showed concentration dependence. Sex 3. adiponectin inhibits the activity of Na+-K+-ATP enzyme on WKY RPT cells, which presents a concentration and dependent.4. as a specificity of the study of the action of adiponectin. Si RNA knocks low Adipo R1 and Adipo R2 in WKY RPT cells. The inhibitory effect of adiponectin on the activity of the enzyme is found to be disappearing. After adding AMPK blockers and E NOS blockers, the inhibitory effect of adiponectin on the activity of Na+-K+-ATP enzyme disappeared. At the same time, adiponectin increased the expression of phosphorylated AMPK and E NOS, and the effect of adiponectin on the enhancement of E NOS phosphorylation was disappearing after the use of AMPK blockers. The diuretic effect of the inhibitory effect of a+-K+-ATP enzyme activity on.6. adiponectin is damaged in SHR. The inhibition of adiponectin to Na+-K+-ATP enzyme is also damaged in SHR RPT cells. The role of adiponectin is supported by the adiponectin receptor agonist Adipo Ron: Adipo Ron has diuretic use, and this effect is in SHR.7.SHR kidney adiponectin. The receptor expression was lower than that of the control WKY rats, but the change in the receptor expression was difficult to explain the loss of function, because the diuretic effect of Adipo R1 and Adipo R2 was still not restored, indicating that there were other factors involved. Our study found that SHR renal adiponectin receptor phosphoric acid phosphate receptor phosphorylation was affected by the phosphorylation of adiponectin receptors. The high phosphorylation level suggests that the level of high phosphorylation may be the cause of the decline in the function of adiponectin receptor.8. to verify whether GRK4 participates in the process of regulating the phosphorylation of adiponectin receptors. We use GRK4? 142V transgenic mice to perfusion the adiponectin receptor agonists and find that the adiponectin receptor participates in the diuretic action of the joint, and is used in the RPT cells of SHR. After GRK4 Si RNA interference, the phosphorylation of adiponectin receptor Adipo R1 and Adipo R2 weakens. This indicates that GRK4 participates in the regulation of phosphorylation of adiponectin receptors, impaired adiponectin excretion and phosphorylation associated with adiponectin receptors in SHR,.9. using microbubbles encapsulated GRK4 Si, and targeted targeting ultrasound in the kidneys. The GRK4 Si RNA released by the broken microbubbles inhibited GRK4, the phosphorylation level of the renal adiponectin receptor weakened, the urine sodium excretion and blood pressure decreased in SHR rats. Meanwhile, the renal function of SHR was not affected, and the renal fibrosis was improved. Conclusion adiponectin has the effect of promoting renal sodium excretion, and the function is impaired in hypertension. It was found that the expression of adiponectin receptor was downregulated, but the receptor did not fully recover the function of adiponectin after the over expression receptor. The phosphorylation of the receptor may play an important role. The increase of GRK4 activity is the cause of the high phosphorylation of adiponectin receptor; UTMD transmission GRK4 Si RNA effectively inhibits the expression of GRK4 in the kidney, and reduces the blood pressure and diuretic sodium in SHR in SHR. Effect.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1

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