Renal Function In Transgenic Mice

转基因小鼠的肾功能

基本信息

批准号：
7967443
负责人：
Jurgen Schnermann
金额：
$ 76.99万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7967443
关键词：
Accounting Adenylate Cyclase Adrenergic Receptor Angiotensin II Angiotensin II Receptor Angiotensin-Converting Enzyme Inhibitors Animals Apical Blood Blood Pressure Blood Vessels Body Fluids Calcium Captopril Cells Conscious Data Diet Distal Distal convoluted renal tubule structure Duct (organ) structure Enalapril Enzyme Inhibitor Drugs Enzyme Inhibitors Epithelial Cells Equilibrium Excretory function Feedback Filtration Furosemide Genes Genotype Glomerular Capillary Goals Golf Hand Immunohistochemistry Individual Intake Juxtaglomerular Cell Kidney Knock-out Label Lead Ligands Limb structure Liquid substance Location Macula densa Maintenance Measures Mediating Messenger RNA Micropuncture Mus NADPH Dehydrogenase Nephrons Nerve Nitric Oxide Synthase Olfactory Epithelium Outcome Pathway interactions Pattern Plasma Play Proteins Regulation Regulatory Pathway Renal function Renin Renin-Angiotensin System Research Role Signal Transduction Sodium Chloride Staining method Stains Surface Sympathetic Nervous System System Tail Testing Thick Time Transgenic Mice Tubular formation Vascular resistance Veins Wild Type Mouse Work absorption beta-adrenergic receptor calbindin capillary bed drinking water falls feeding glomerular filtration improved indexing inhibitor/antagonist kidney cortex kidney medulla mouse model pressure prevent promoter receptor recombinase research study response salt balance salt intake

项目摘要

1. Components of the olfactory chemosensory signaling system have previously been found outside of the olfactory epithelium. Using immunohistochemistry we have confirmed that adenylyl cyclase 3 (AC3) and the universal olfactory g protein (Golf ) are expressed in the kidney cortex as well as in the renal medulla. The cortical Golf labeling was associated with the distal convoluted tubule (DCT) as shown by colocalization in serial sections with the NaCl cotransporter NCC, a highly specific marker of the DCT. Golf staining also co-localized with calbindin indicating that it is expressed along the connecting tubule and collecting duct. There was no Golf staining in the thick ascending limb. The localization of AC3 in the kidney had a pattern of expression very similar to that of Golf. AC3 clearly stained distal tubules typically along the apical surfaces of the tubular epithelial cells, and it showed colocalization with NCC in serial sections, but not with a marker of the thick ascending limb. Interestingly, Golf and AC3 were also expressed in the macula densa (MD), a location that was confirmed by overlap with NADPH diaphorase staining, a positive marker of macula densa cells on account of the very specific expression of NO synthase in these cells. The localization of AC3 in macula densa cells suggested that the olfactory system may play a role in specific macula densa functions such as control of vascular tone through the tubuloglomerular feedback mechanism and renin secretion. To assess the TGF profile in wild type and AC3-deficient mice, we performed micropuncture experiments in which the flow rate in the distal segment of a nephron was manipulated while the proximal stop-flow pressure (PSF) in that same nephron was measured as an index of glomerular capillary pressure. Under these experimental conditions TGF response magnitude was not measurably different between wild type and AC3−/− mice. Furthermore, flow rates inducing half-maximal responses were statistically indistinguishable between genotypes (9.6 nL/min in wild type and 9.5 nL/min in AC3−/− mice). Arterial blood pressure at the time of micropuncture was similar between wild type and AC3−/− mice. On the other hand, plasma levels of renin in conscious AC3+/+ and AC3−/− mice were about 50% lower in AC3−/− mice as compared with those found in their wild type littermates. Thus, although the presence of AC3 and Golf remains highly intriguing their functional role is not entirely clear. 2. The renin-angiotensin system plays a critical role in the maintenance of body salt balance by regulating blood pressure and NaCl excretion by the kidneys. It is therefore not surprising that renin secretion and subsequently plasma renin concentration are changing when NaCl intake changes. In a very predictable manner renin secretion falls in states of NaCl excess and it increases when NaCl intake falls. Despite this well established phenomenon the mechanisms responsible for salt-induced regulation of renin secretion are still unclear. Since the changes in body fluid volume resulting from alterations in NaCl intake lead to changes in sympathetic renal nerve activity and since it is known that renal sympathetic nerves exert pronounced effects on renin secretion through adrenergic -receptors, we assessed the role of -adrenergic receptors in salt-dependent regulation of renin release by determining the effect of salt intake on plasma renin concentration (PRC, ng Ang I/ml hr) and renin mRNA in conscious wild type and 1/2-adrenergic receptor-deficient mice (1/2 ADR-/-). Plasma renin concentration was determined in tail vein blood as measure of renin secretion. On a control diet, plasma renin concentration (ng AngI/ml hr) was significantly higher in wild type than 1/2 ADR-/- mice (1338395 vs. 30448; p=.04). Plasma renin concentration of mice kept on a low Na diet (.003%) for one week increased significantly in both wild type and 1/2 ADR-/- mice (to 2789555 and 73354 respectively). Similarly, a high Na diet (8%) suppressed PRC in both genotypes (to 676213 in wild type, and to 8524 in 1/2 ADR-/-). In additional studies we observed that during inhibition of macula densa NaCl transport by furosemide plasma renin increased in both wild type and 1/2 ADR-/- mice (to 70841073 and to 3277154 respectively). The increment of plasma renin caused by furosemide was augmented by a low Na diet and diminished by a high Na diet in both wild type and 1/2 ADR-/- mice. These data indicate that renin synthesis and release under basal conditions is markedly dependent upon the presence of beta-adrenergic receptors, that the modulating effect of salt intake is maintained in the absence of beta-adrenergic receptors, and that the stimulatory effect of furosemide is maintained in the absence of beta-adrenergic receptors. Thus, it appears that regulation of renin release by salt diet and furosemide is dominated by regulatory inputs other than the sympathetic nervous system. 3. Angiotensin II has long been known to inhibit renin secretion, an outcome thought to be mediated by angiotensin II receptors (AT1a) on the renin-secreting juxtaglomerular cells and termed short feedback loop inhibition of renin release. Conversely, the logical explanation for the stimulatory effect of angiotensin II converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) has been escape from short loop feedback inhibition by angiotensin II. The renin-inhibitory effect of angiotensin II at the cellular level is mediated by the increase in cytosolic calcium caused by stimulation of the Gq-coupled AT1 receptor implying that ACEI and ARB may work through reductions in cell Ca. In the current experiments we examined the hypothesis that angiotensin II blockade acts predominantly through Gs-mediated stimulation of adenylyl cyclase (AC) by testing the effect of ACEI and ARB in mice with juxtaglomerular cell-specific deficiency in the AC-stimulatory Gs generated by conditional knockout of a floxed Gs gene with cre recombinase driven by the endogenous renin promoter. This mouse model has previously been used to selectively delete Gs from JG cells. The ACEI captopril and quinaprilate, and the ARB candesartan significantly increased plasma renin concentration (PRC) to 20-40 times basal plasma renin in wild type mice, but did not significantly alter plasma renin in Gs-deficient mice. In mice fed a low NaCl diet (0.03%) for 7 days and receiving the ACE inhibitor enalapril via the drinking water, plasma renin increased 35 fold (n=12, p<0.001) in wild type mice, whereas it did not change significantly in the Gs-deficient animals (n=12, p=0.17). Pharmacological inhibition of adenylyl cyclase with three different inhibitors reduced the stimulatory effect of captopril by 70-80%. We conclude that the stimulatory effects of angiotensin II blockade on renin synthesis and release are for the most part indirectly mediated by the action of ligand(s) that utilize Gs -dependent pathways.

1。嗅觉化学感信号系统的成分先前已在嗅觉上皮之外发现。使用免疫组织化学我们已经证实，腺苷酸环化酶3（AC3）和通用嗅觉G蛋白（高尔夫）在肾脏皮质以及肾脏髓质中表达。皮质高尔夫标记与远端曲折小管（DCT）相关，如串行切片与NaCl Cotransporter NCC的共定位所示，DCT的高度特异性标记。高尔夫染色也与卡尔比丁共定位，表明它沿着连接小管和收集管道表达。厚厚的肢体没有高尔夫染色。 AC3在肾脏中的定位具有与高尔夫的表达方式。 AC3显然染色的远端小管通常沿着管状上皮细胞的顶端表面，并且在串行切片中与NCC进行了共定位，但没有与较厚的上升肢的标记。有趣的是，高尔夫和AC3也在黄斑densa（MD）中表达，该位置通过与NADPH diaphorase染色重叠证实，这是Macula densa细胞的阳性标记，这是由于这些细胞中无合酶非常具体的表达。 AC3在黄斑densa细胞中的定位表明，嗅觉系统可能在特定的黄斑densa函数中起作用，例如通过肾小球胶质细胞反馈机制和肾素分泌控制血管张力。为了评估野生型和AC3缺陷型小鼠的TGF曲线，我们进行了微功能实验，其中操纵了肾单位的远端段中的流速，而在同一肾单位的近端停止水压（PSF）则以肾小球胶合压力的指数测量。在这些实验条件下，野生型和AC3 - / - 小鼠之间的TGF响应幅度并没有差异。此外，在基因型之间诱导一半最大响应的流速在统计学上是无法区分的（野生型为9.6 nl/min，AC3 - / - 小鼠中的9.5 nl/min无法区分。微呼吸时的动脉血压在野生型和AC3 - / - 小鼠之间相似。另一方面，与在野生型同窝仔相比，AC3 - / - 小鼠中有意识的AC3+/+和AC3 - / - 小鼠中肾素的等离子水平低约50％。因此，尽管AC3和高尔夫的存在仍然高度吸引人的功能作用并不完全清楚。 2。肾素 - 血管紧张素系统通过调节肾脏的血压和NaCl排泄来维持体内盐平衡的作用至关重要。因此，当NaCl摄入发生变化时，肾素分泌和随后的血浆肾素浓度正在发生变化也就不足为奇了。肾素分泌以非常可预测的方式落在NaCl过量状态下，当NaCl摄入量下降时，它会增加。尽管这种良好的现象仍然尚不清楚负责盐诱导的肾素分泌调节的机制。 Since the changes in body fluid volume resulting from alterations in NaCl intake lead to changes in sympathetic renal nerve activity and since it is known that renal sympathetic nerves exert pronounced effects on renin secretion through adrenergic -receptors, we assessed the role of -adrenergic receptors in salt-dependent regulation of renin release by determining the effect of salt intake on plasma renin concentration (PRC, ng Ang I/ml HR）和肾素mRNA中有意识的野生型和1/2-肾上腺素能受体缺陷型小鼠（1/2 ADR - / - ）。血浆肾素浓度在尾静脉血液中确定为肾素分泌的度量。在对照饮食中，野生型的血浆肾素浓度（NG ANGI/ML HR）明显高于1/2 ADR - / - 小鼠（1338395 vs. 30448; p = .04）。在野生型和1/2 ADR - / - 小鼠（分别为278955555和73354）中，小鼠的血浆肾素浓度保持低Na饮食（.003％）。同样，高Na饮食（8％）在两种基因型中抑制了PRC（在野生型中为676213，在1/2 ADR - / - 中抑制了8524。在其他研究中，我们观察到，在野生型和1/2 adr - / - 小鼠中抑制黄斑densa NaCl转运期间（分别为70841073和3277154）。低Na饮食会增强由速尿引起的血浆肾素的增加，并通过野生型和1/2 ADR - / - 小鼠的高Na饮食减少。这些数据表明，在基础条件下的肾素合成和释放明显取决于β-肾上腺素能受体的存在，即在没有β-肾上腺素能受体的情况下保持盐摄入的调节作用，并且在不存在β-肾上腺素能受体的情况下，速尿的刺激作用是维持的。因此，看来通过盐饮食和速尿对肾素释放的调节是由除交感神经系统以外的调节输入所支配的。 3.血管紧张素II长期以来众所周知会抑制肾素分泌，这一结果被认为是由血管紧张素II受体（AT1A）介导的，对肾素分泌近去脂肪胶质细胞的介导，并被称为短期反馈循环抑制肾素释放。相反，血管紧张素II转化酶抑制剂（ACEI）或血管紧张素II受体阻滞剂（ARB）的刺激作用的逻辑解释已从血管紧张素II的短环反馈抑制中逃脱。血管紧张素II在细胞水平上的肾素抑制作用是由刺激GQ耦合AT1受体引起的胞质钙的增加所介导的，这表明ACEI和ARB可能通过降低细胞Ca而起作用。在当前的实验中，我们检查了以下假说：血管紧张素II阻断主要通过GS-介导的腺苷环酶（AC）刺激通过测试ACEI和ARB在小鼠中的ACEI和ARB在与AC刺激性GN中的AC刺激性GS中产生的AC型rene的脱骨剂的脱骨剂的脱骨剂的脱孔剂的含量不足的小鼠中的小鼠对CRE脱落的cre gene的最终结构的脱发，而cre gene则是cre gene的最终组合的含量。发起人。该小鼠模型先前已被用于从JG细胞中选择性删除GS。 ACEI CATCEROPRIL和QUINAPRATE，ARB Candesartan在野生型小鼠中显着将血浆肾素浓度（PRC）显着提高至20-40倍的基础血浆肾素，但并未显着改变GS缺乏小鼠的血浆肾素。在喂养低NaCl饮食（0.03％）的小鼠中，持续7天，并通过饮用水接收ACE抑制剂依那倍lil，血浆肾素在野生型小鼠中增加了35倍（n = 12，p <0.001），而在GS缺乏的动物中没有显着变化（n = 12，p = 0.17）。用三种不同抑制剂对腺苷酸环化酶的药理抑制作用将卡托普利的刺激作用降低了70-80％。我们得出的结论是，血管紧张素II阻断对肾素合成和释放的刺激作用在很大程度上是通过利用GS依赖性途径的配体的作用而间接介导的。