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胃泌素通过诱导肾脏摄取L-DOPA增加多巴胺合成参与血压调节
【作者】 姜晓亮；
【作者基本信息】 北京协和医学院 ， 病理学及病理生理学， 2014， 博士
【摘要】 第一部分：胃泌素通过诱导肾脏摄取L-DOPA增加多巴胺合成参与血压调节背景及目的原发性高血压(Essential Hypertension, EH)是一种严重危害人类健康的多因素疾病,其发病趋势逐年上升。动脉血压不仅决定于血管阻力,也决定于心输出量。心输出量受到细胞外液钠容量,肾功能等因素的影响,总外周阻力则受交感神经系统、儿茶酚胺等激素的影响。新近研究发现,胄泌素(多肽类激素)不仅能够调节胃酸分泌,而且,对体内钠离子平衡有重要的调节作用。而多巴胺在钠水代谢及血压调节方面的重要作用已被业界公认。所有多巴胺受体亚型均直接或间接地通过与其他血压调节系统交互作用,参与肾脏钠排泄和血压的调节。本文就胃泌素与多巴胺相互作用的可能机制进行探索研究,从全新的视角阐述其在血压调控中的作用,为原发性高血压的防治提供新靶点。方法于安徽省阜阳市人民医院对高血压人群(HT,n=95)和正常人群(NT,n=82)进行研究。分光法检测空腹状态下血生化指标。放射免疫法检测空腹和餐后30,60,120分钟血清胃泌素以及空腹肾素,血管紧张素和醛固酮激素含量变化情况并监测各个时间点的血压变化。通过相关性分析基础状态下胃泌素与肾素,血管紧张素和醛固酮激素的相关性。于培养有小鼠和人肾脏近曲小管细胞(Mouse proximal tubular cells, MPTCs and Human proximal tubular cells, HPTCs)的培养基中加入100uM的L-DOPA,同时加入胃泌素,并且设定两个浓度50ng/ml和100ng/ml, ELISA法检测生成多巴胺的量,判断胃泌素的最佳用药浓度。在细胞培养基中加入L365,260(CCKB拮抗剂)或BCH(L-氨基酸转运体抑制剂)预处理6分钟,而后加入100uM的L-DOPA和100ng/ml胃泌素,检测生成多巴胺的量。BCA法检测细胞蛋白浓度。Western blotting方法检测药物处理细胞LAT1总蛋白和膜蛋白的表达情况。将新鲜小鼠肾脏切碎加入细胞培养基中培养采用同浓度的L-DOPA(?)胃泌素处理,检测组织生成的多巴胺的量。Western blotting方法检测药物处理小鼠肾脏组织LAT1总蛋白和膜蛋白的表达情况。无菌制备胃泌素siRNA,为单侧肾脏切除的Balb-C小鼠植入迷你泵,连续释放胃泌素siRNA,沉默胃泌素基因。股动脉插管测定胃泌素siRNA和mock siRNA注射前后的动脉血压。Western blotting方法检测肾脏组织胃泌素蛋白表达,PCR方法检测胃泌素mRNA的表达情况。Western blotting方法检测胃泌素siRNA和mock siRNA处理小鼠肾脏组织LAT1总蛋白和膜蛋白的表达情况。结果人群实验显示胃泌素与高血压密切相关。HT组和NT组空腹胃泌素水平无明显差异,但HT组餐前血压显著高于正常组(P<0.05)。餐后HT组血压显著降低但120分钟后并未恢复且持续降低差异显著(P<0.05),而正常组餐后血压较餐前并无显著性降。血清胃泌素监测显示HT组餐后血清胃泌素变化趋势与对照组相似,即30分钟时胃泌素水平达到峰值随后逐渐降低。但HT组血清胃泌素水平在餐后30,60,和120分钟显著高于正常组(P<0.05)空腹状态下HT组胃泌素水平与醛固酮和血管紧张素Ⅱ有显著相关性(P<0.05)。基础研究说明胃泌素能够使小鼠和人源肾脏近曲小管细胞摄取更多的L-DOPA,生成多巴胺增多。L365,260预处理细胞结果证实了胃泌素刺激多巴胺的生成是通过作用于CCKBR而发牛。L-氨基酸转运体抑制剂BCH能够使L-DOPA摄取显著降低(P<0.01),证实了钠离子依赖型L-氨基酸转运体在胃泌素诱导的L-DOPA摄取中起主导作用。Western blotting检测结果显示胄泌素诱导机制为,够刺激细胞膜LAT1数量增多,而非总LAT1表达增加。MPTCs摄取L-DOPA的量显著低于HPTCs(12±1.5vs28±1.9P<0.05)。造成这一结果的原因可能是小鼠细胞来源于C57BL/6J,该小鼠可能存在L-DOPA摄取障碍。离体实验同样证实了胃泌素对于体内多巴胺的正常合成和血压起到非常重要的作用。结论我们首次通过流行病调查的方法显示了高血压人群餐后胃泌素显著升高,并且证实空腹状态下胃泌素与血管紧张素Ⅱ和醛固酮有显著相关性,证实了胃泌素与血压调节密切相关。体外实验、动物实验和离体实验都证实了胄泌素能够通过CCKBR诱导摄取L-DOPA增多,导致多巴胺生成增多,其氨基酸转运机制主要通过LAT1。第二部分：多巴胺D2受体基因单核苷酸多态性诱导人近曲小管细胞发生炎症反应和纤维化背景及目的多巴胺D2受体(D2R)能够反向调节小鼠肾脏近曲小管细胞炎症反应,小鼠D2R功能障碍更容易发生肾脏炎症反应以及纤维化损伤。一些常见的人(h)DRD2基因单核苷酸多态性位点(SNPs; rs6276,6277, and1800497)与D2R表达和功能以及血压调节密切相关,但其是否参于肾脏炎症反应和纤维化并不清楚。我们研究人肾脏近曲小管细胞(HPTCs)携带这些D2R SNPs对十肾脏炎症反应和纤维化损伤的影响,为慢性肾脏疾病和高血压的基因治疗提供新靶点。方法Western blotting和Real-time PCR方法检测携带D2R SNPs HPTCs和对照组细胞的D2R蛋白和mRNA的表达情况。D2R激动剂刺激细胞后,通过化学发光免疫测定法检测cAMP的生成量,从而观察D2R的功能状态。相同方法方法检测携带D2R SNPs HPTCs和对照组细胞的促炎因子TNFa蛋白和mRNA的表达量,检测细胞因子和趋化因子的表达量。同样通过Western blotting和Real-time PCR方法检测携带D2R SNPs HPTCs和对照组细胞的促纤维化因子TGFβ1蛋白和mRNA的表达量,以及其信号通路下游因子的表达情况,比如说SMAD3、Snail1、FN-1、Col1a和Vimentin。免疫荧光法检测TGFβ1和下游因子的表达情况,观察细胞形态变化情况。逆向实验向携带D2R SNPs HPTCs瞬时转染人DRD2基因,对照组转染空载体,使得细胞表达足够的D2R。Western blotting和Real-time PCR方法检测转染后细胞D2R蛋白和mRNA的表达情况。Real-time PCR方法检测转染后细胞TGFβ1和Snail1、FN-1、Col1a和Vimentin mRNA的表达量。并且检测TGFβ1和FN-1蛋白表达量。免疫荧光法检测瞬时转染人DRD2基因后TGFβ1和下游因子的表达情况,观察细胞形态变化情况。结果HPTCs携带D2R SNPs与对照组细胞相比较D2R蛋白和mRNA的表达显著降低,功能显著减弱(P<0.05, t-test)。HPTCs携带D2R SNPs与对照组细胞相比较促炎因子TNFα、细胞因子、趋化因子和促纤维化因子TGFβ1以及下游信号靶蛋白Smad3和Snail1的表达显著增加(P<0.05, t-test),出现上皮细胞-间质细胞转化(EMT),并且产生大量细胞外基质蛋白(FN-1、Col1a和Vimentin的表达量增多)。结果表明RPTCs携带D2R SNPs的D2R的表达和功能降低诱导细胞促炎因子和促纤维化因子分泌增加,EMT标志因子分泌增多。为检测D2R SNPs的特异性,向携带D2R SNPs的HPTCs瞬时转染含人DRD2基因的质粒后发现D2R的表达显著升高但并无过表达现象。与对照组相比D2R表达升高诱导TGFβ1, Smad3、Snail1表达均显著降低(P<0.05, t-test),细胞外基质蛋白(FN-1、Col1a和Vimentin)减少。免疫荧光结果也显示细胞出现EMT现象,形状逐渐变为长梭形。结果再次证实了携带有D2R SNPs的人D2R表达和功能均低于正常人,因而更有可能发展肾损伤和肾脏纤维化。结论实验结果表明,D2R有保护人肾脏近曲小管的功能,可以降低炎症因子、促纤维化因子表达,减少EMT标志因子的分泌。但当人体携带有这些D2R SNPs时可能会促进肾脏炎症反应和肾脏纤维化的发展。通过基因检测早发现体内存在的患病风险,早期通过药物干预改善肾纤维化和慢性肾脏病的发展。
【Abstract】 Gastrin increases renal dopamine production by increasing the uptake of L-DOPA which play an important part in the regulation of blood pressureObjective Essential hypertension (EH) is a multi-factor disease, which is a danger to human health and the incidence trend is rising year by year. Arterial blood pressure not only depends on the vascular resistance, but also is determined by the cardiac output. Cardiac output is affected by sodium extracellular fluid volume and renal function. The total peripheral resistance is affected by the influence of the sympathetic nervous system and catecholamine hormone. Recent study found that Gastrin is a peptide hormone, which acts not only to regulate gastric acid secretion, but also to exert physiological actions such as the regulation of sodium balance. While many studies suggest that dopamine also play an important role in sodium excretion and blood pressure regulation. Dopamine receptor subtypes all directly or indirectly, interact with other blood pressure regulating system, and are involved in the regulation of renal sodium excretion and blood pressure. This article gives a possible mechanism of the interaction between gastrin and dopamine, which shows a new perspective on blood pressure regulation and provides a new target for prevention and treatment.Methods By a case (HT, n=95)-control (NT, n=82) study in Fuyang People’s Hospital, Anhui Province, China, Basic information on the subjects was collected. Blood samples were collected, and blood pressures were measured at30,60, and120min after the meal ingestion. Correlation between basal serum levels of gastrin and other hormones was calculated. In vitro study, renal proximal tubular cells were incubated with L-DOPA(100uM) and separated concentration of gastrin (50ng/ml or lOOng/ml) applied from the cell side, ELISA kit was used to measure dopamine production. Cells were pre-incubated for6min with a single concentration (1μM) of L365,260or BCH (lmmol/L) before L-DOPA (100uM) and gastrin (100ng/ml) treatment to measure dopamine production. Protein concentration was detected by BCA protein assay kit. Western blotting was performed to detect the expression of whole and plasma membrane LATl expression of cells after drug treatment. Fresh kidney pieces were cultured in DMEM medium with drug treatment as same as in vitro study. Western blotting was performed to detect the expression of whole and plasma membrane LAT1expression of tissure. The Gastrin-specific siRNA was prepared in an in vivo transfection reagent (Mirus Bio) under sterile conditions. A mini-osmotic pump was inserted and positioned subcapsularly in Uninephrectomized adult male BALB/cJ mice to continuously deliver the Gastrin-specific siRNA and silencing gastrin expression. The blood pressure was measured from femoral artery of mice before and after mock or gastrin-specific siRNA infusion. Western blotting was performed to detect gastrin expression.gastrin mRNA was measured by Real-time PCR. Western blotting was performed to detect the expression of whole and plasma membrane LAT1expression of mice kidney treated by gastrin siRNA and mock siRNA.Results Clinical study suggests that gastrin is involved in the regulation of blood pressure. There is also no difference in basal serum gastrin levels between the two groups, but fasting blood pressures are significantly higher in HT than NT (P<0.05). In the HT group, the blood pressure significantly decreases after the meal and remains low level, Food intake does not change the blood pressure in NT. The fasting and pattern of the postprandial increase (peak at30min) in serum gastrin levels are similar in HT and NT. However, the gastrin levels at30,60, and120min are significantly higher in HT than NT (P<0.05, t test). Fasting serum levels of gastrin are significantly correlated with fasting serum levels of aldosterone and angiotensin Ⅱ (P<0.05, Pearson) in HT but not NT. Mechanism research shows that gastrin can stimulate mouse proximal tubular cells(MPTCs) and human proximal tubular cells (HPTCs) uptake more L-DOPA from extracellular. Pre-incubation of L365,260decrease dopamine production indicating that gastrin via CCKBR stimulates uptake of L-DOPA. L-amino acid transporter plays an important role in the uptake of L-DOPA by treatment of BCH. Western blotting shows that gastirn can increase the number of LAT1on plasma membrane, but not the whole LAT1expression.We also show that uptake of L-DOPA in MPTCs is significantly lower than in HPTCs(12±1.5vs28±1.9P<0.05), maybe due to the stains of mice (C57BL/6J). Ex vivo study also demonstrates that gastrin plays an important role in maintaining normal dopamine synthetic and blood pressure.Conclution Based on our findings that postprandial serum gastrin levels are significantly higher in hypertensive than normaltensive, and fasting serum levels of gastrin are significantly correlated with fasting serum levels of aldosterone and angiotensin II, we conclude that gastrin is involved in the regulation of blood pressure. In vivo, in vitro and ex vivo studies indicate that gastrin can increase dopamine production by uptake of L-DOPA, in whcih LAT1may participate. Single nucleotide polymorphisms of the dopamine D2receptor increase inflammation and fibrosis in human renal proximal tubule cellsObjective The dopamine D2receptor (D2R) negatively regulates inflammation in mouse renal proximal tubule cells (RPTCs) and lack or downregulation of the receptor in mice increases the vulnerability to renal inflammation independent of blood pressure. Some common single nucleotide polymorphisms (SNPs; rs6276,6277, and1800497) in the human (h) DRD2gene are associated with decreased D2R expression and function, as well as high blood pressure. We tested the hypothesis that human RPTCs expressing these SNPs have increased expression of inflammatory and injury markers, which could provide clues for the gene therapy of chronic renal disease and high blood pressure.Method Western blotting and Real-time PCR weres performed to detect the protein and mRNA expression of D2R in hRPTCs carrying D2R SNPs and cells carrying no D2R SNPs. After D2R agonist treatment, cyclic AMP accumulation was tested by cAMP chemiluminescent immunoassay, which results effect D2R function. We then measure the protein and mRNA expression of pro-inflammatory TNFa in hRPTCs carrying D2R SNPs and cells carrying no D2R SNPs. Real-time PCR was also used to test mRNA expression of cytokines/chemokines.The protein and mRNA expression of TGFβ1its signaling pathway facrors SMAD3, Snail1, FN-1, Col la and Vimentin were measured by Western blotting and Real-time PCR. Immunofluorescence was performed to detect TGFβ1and its signaling target, and cellular morphology. We reverse the experiment by transfected with an plasmid harboring wild-type human DRD2or empty vector in hRPTCs carrying SNPs, to make the cells have enough D2R expression. Western blotting and Real-time PCR were performed to detect the protein and mRNA expression of D2R, TGFβ1and Snail1, FN-1, Col la and Vimentin in Immunofluorescence was performed to detect TGFβ1and its signaling target, and cellular morphology, which is same as non-transfected cells. Result Human RPTCs with D2R SNPs had decreased D2R expression and function comparing with cells carrying no D2R SNPs (P<0.05, t-test). The expressions of the pro-inflammatory TNFa and the pro-fibrotic TGFβ1and its signaling targets Smad3and Snail1were increased (P<0.05, t-test) in hRPTC with D2R SNPs. These cells also showed induction of epithelial mesenchymal transition (EMT) and production of extracellular matrix proteins, assessed by increased vimentin, fibronectin-1(FN-1), and Col1a. Our results show that RPTCs from subjects carrying D2R SNPs that result in decreased D2R mRNA and protein express a pro-inflammatory and pro-fibrotic phenotype and markers of EMT. To test the specificity of these D2R SNP effects, hRPTC with D2R SNPs were transfected with a plasmid encoding wild-type DRD2. D2R expression was increased and those of TGFβ1, Smad3, Snail1, vimentin, fibronecti-1and Col1a were decreased (P<0.05, t-test) in hRPTC with D2R SNPs transfected with wild-type DRD2compared to hRPTC-D2R SNP transfected with empty vector. Result of Immunofluorescence shows the change of cellular morphology, cells became spindle and disorder. The result demonstrated again individuals carrying D2R polymorphisms that result in decreased D2R function could be more vulnerable to renal injury and fibrosis.Conclusion These data support the hypothesis that D2R function has protective effects in human RPTCs, which can decrease expression of pro-inflammatory and pro-fibrotic phenotype and markers of EMT. This study suggests that carriers of these SNPs may be prone to chronic renal disease and high blood pressure. Genetic testing could identify the individuals at risk and pharmacological treatment tailored to ameliorate the insult which should result in decreased prevalence of renal injury and chronic kidney disease.