Renal Function In Transgenic Mice

转基因小鼠的肾功能

基本信息

批准号：
8553484
负责人：
Jurgen Schnermann
金额：
$ 93.8万
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/8553484
关键词：
AQP1 gene Address Adenosine Agonist Angiotensin II Angiotensin II Receptor Animals Blood Pressure Blood Vessels Body Fluids Captopril Cells Characteristics Clinic Communication Coupled Coupling Cultured Cells Enzyme Inhibitor Drugs Enzyme Inhibitors Epithelium Equilibrative Nucleoside Transporter 1 Equilibrium Excretory function Feedback Filtration GTP-Binding Protein alpha Subunits, Gs Gap Junctions Genes Genetic Genetic Recombination Glomerular Capillary Goals Incidental Findings Individual Inosine Juxtaglomerular Apparatus Juxtaglomerular Cell Kidney Ligands Link Liquid substance Macula densa Maintenance Measures Mediating Membrane Motor Activity Movement Mus Nucleosides Outcome Pathway interactions Permeability Plasma Play Proteins Proximal Kidney Tubules Regulation Regulatory Pathway Renal function Renin Research Residual state Role Route Running Signal Transduction Site Smooth Muscle Actin Staining Method Smooth Muscle Myocytes Sodium Chloride Structure of glomerular mesangium Tight Junctions Tissues Transgenic Mice Translation Alteration Tubular formation Vascular resistance Vasoconstrictor Agents Water Wild Type Mouse absorption arteriole capillary bed connexin 40 extracellular glomerular filtration improved mesangial cell nestin protein nucleotide metabolism pressure prevent promoter recombinase response salt intake sensor solute carrier family 29 (nucleoside transporters), member 1 protein, mouse transmission process uptake

项目摘要

1. To address the question whether selective deletion of A1AR from smooth muscle cells is sufficient to abolish TGF responsiveness we have determined TGF response magnitude in mice in which vascular A1AR deletion was achieved using the loxP recombination approach with cre recombinase being controlled by a smooth muscle actin promoter (SmCre/A1ARff). Effective vascular deletion of A1AR was affirmed by absence of vasoconstrictor responses to adenosine or the A1AR agonist cyclohexyl adenosine (CHA) in microperfused afferent arterioles. TGF responses in SmCre/A1ARff mice were found to be significantly reduced, but not abolished. Maintenance of residual TGF activity despite absence of A1AR-mediated responses in AA suggests participation of extravascular A1AR in TGF. Support for this notion comes from the observation that deletion of A1ARff by nestin-driven cre recombinase caused an identical TGF response reduction while arteriolar responsiveness to A1AR activation was reduced, but not abolished. A1AR on arteriolar smooth muscle cells are primarily responsible for TGF activation, but A1AR on extravascular cells, perhaps mesangial cells, appear to contribute to the TGF response. 2. While previous evidence has indicated that the A1AR ligand adenosine is at least in part derived from the extracellular metabolism of nucleotides transmembrane movement of adenosine mediated by the widely distributed equilibrative nucleoside transporter 1 (ENT1) may also play a modifying role. To study this possibility we have assessed JGA function in ENT1-deficient mice (breeder pairs of ENT1-/- mice were kindly provided by Doo-Sup Choi, Mayo Clinic, Rochester, MN). We observed that TGF responses were significantly reduced in ENT1-/- compared to WT mice and that this was independent of genetic background. This observation points to a role of nucleoside transport in the NaCl-synchronous changes of extracellular adenosine levels. Since plasma concentrations of adenosine and of its breakdown product inosine were markedly higher in ENT1-/- than in WT mice one may assume that the direction of adenosine transport in most tissues consists of cellular uptake. Our observations therefore indicate that interference with adenosine disposition by cellular uptake may modify TGF characteristics, perhaps by preventing the translation of alterations of luminal NaCl concentrations into synchronous fluctuations of adenosine levels. An incidental finding of these studies was that spontaneous locomotor activity levels as well as voluntary wheel running activity was significantly reduced in ENT1-/- animals, probably resulting from the central inhibitory effects of the elevated tissue adenosine levels. 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. Therefore, the explanation for the stimulatory effect of the clinically widely used angiotensin II converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) has been escape from short loop feedback inhibition. Using mice with conditional deletion of the Gs alpha gene in renin-producing cells we found that the ACEI captopril and quinaprilate, and the ARB candesartan did not significantly alter plasma renin although these compounds increased plasma renin concentration (PRC) more than 20fold in wild type mice. Since the stimulatory effect of angiotensin II is Gs alpha-dependent, and since angiotensin II receptors are not coupled to Gs alphaz one may conclude that the stimulatory effect of angiotensin II blockade on renin synthesis and release is indirectly mediated by the action of ligand(s) that utilize Gs alpha-dependent pathways. 4. Gap junctions provide a pathway for the functional coupling of individual cells, and such coupling may be important in the communication mechanisms that link the different cells of the juxtaglomerular apparatus. In fact, the gap junctional protein connexin 40 (Cx40) is highly expressed in the extraglomerular mesangium connecting the tubular epithelium at the macula densa and the afferent arteriole. Participation of Cx40 in juxtaglomerular communication is suggested by the finding that the expression of renin is increased and the localization of renin-expressing cells is abnormal in Cx40-deficient mice. Furthermore, in Cx40-deficient mice on two different genetic backgrounds TGF responsiveness was found to be diminished in magnitude and slowed in response velocity suggesting that gap junctional communication may contribute to signal transmission. 5. Fluid reabsorption in tubular segments upstream of the macula densa is part of the JGA-dependent regulation since it determines the NaCl load delivered to the sensing site. We have previously shown that aquaporin 1 in the proximal tubule is required for the high rate of absorption by providing transmembrane entry and exit pathways for water. However, it has remained unclear if water in the proximal tubule can also cross the epithelium through a paracellular pathway. The identification of claudin2 (cldn2) as a tight junction protein highly expressed in renal proximal tubules together with evidence that cldn2 increases water permeability in cultured cells has prompted us to compare proximal fluid reabsorption in wild type and cldn2-deficient mice. We found that cldn2-dependent fluid reabsorption amounted to a fraction of 27.5% of total reabsorption suggesting a sizable paracellular route of proximal fluid transport. Reduced fluid reabsorption in cldn2-deficient mice was associated with a reduced GFR, perhaps a consequence of increased salt delivery to the TGF sensor. We are currently generating mice with deletions of both aquaporin 1 and claudin 2 to examine if the components of fluid reabsorption mediated by membrane or tight junction proteins are independent and additive.

1。为了解决一个问题，是否从平滑肌细胞中选择性删除A1AR是否足以消除TGF响应能力，我们已经确定了小鼠中TGF响应幅度，在这种小鼠中，使用LOXP重新组合方法可以通过CRE重组酶来控制平滑肌肌肉肌动蛋白（SMCRE/smcre/a1ar a1ar a1ar a1ar a1ar a1ar a1ar a1ar a1ar a1ar）。由于没有血管收缩反应或在微填充传入小动脉中的A1AR激动剂环己糖苷（CHA），A1AR的有效血管缺失得到了肯定。发现SMCRE/A1ARFF小鼠中的TGF反应显着降低但未被废除。尽管没有A1AR介导的反应，但仍在维持剩余的TGF活性，这表明血管外A1AR参与TGF。对这一概念的支持来自于观察到，巢蛋白驱动的CRE重组酶对A1ARFF的缺失导致了相同的TGF响应降低，而小动脉对A1AR激活的反应性降低了，但没有被废除。小动脉平滑肌细胞上的A1AR主要负责TGF激活，但是血管外细胞上的A1AR（也许是肾小球细胞）似乎有助于TGF反应。 2。尽管以前的证据表明，A1AR配体腺苷至少部分源自核苷酸的细胞外代谢，该核苷酸的核苷酸跨膜跨膜运动的腺苷移动由广泛分布的平衡核苷转运蛋白1（ENT1）介导的腺苷运动也可能起作用。为了研究这种可能性，我们已经评估了ENT1缺陷型小鼠的JGA功能（ENT1 - / - 小鼠的育种对由Mayo Chair，Mayo Clinic，Mayo Clinic，Mayo clinics and and）。我们观察到与WT小鼠相比，ENT1/ - 的TGF响应显着降低，这与遗传背景无关。该观察结果表明核苷转运在细胞外腺苷水平的NaCl同步变化中的作用。由于ENT1 - / - 的血浆浓度及其分解产物的肌苷明显高于WT小鼠，因此可能假设大多数组织中腺苷转运的方向由细胞摄取组成。因此，我们的观察结果表明，通过细胞摄取对腺苷的分配的干扰可能会改变TGF特征，也许是通过防止向腔内NaCl浓度转化为腺苷水平同步波动的变化。这些研究的一个偶然发现是，ENT1 - / - 动物的自发运动活性水平以及自愿性轮跑活动显着降低，这可能是由于升高的组织腺苷水平的中心抑制作用而产生的。 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。因此，对临床广泛使用的血管紧张素II转化酶抑制剂（ACEI）或血管紧张素II受体阻滞剂（ARB）的刺激作用的解释已从短环反馈抑制中逃脱。使用与肾素产生细胞中GS Alpha基因有条件缺失的小鼠，我们发现ACEI Captopril和Quinaprilate和ARB Candasartan并未显着改变血浆肾素，尽管这些化合物增加了血浆肾素浓度（PRC）在野生型小鼠中的20倍数超过20倍。由于血管紧张素II的刺激作用是GSα依赖性的，并且由于血管紧张素II受体与GS Alphaz偶联，因此可以得出结论，即血管紧张素II阻断对肾素合成和释放的刺激作用是通过使用Ligand（S）的作用而介导的，该作用是通过使用gs alpha依赖性path的配体的作用来介导的。 4。间隙连接为单个细胞的功能耦合提供了途径，并且这种耦合在连接近胶质体设备的不同细胞的通信机制中可能很重要。实际上，间隙连接蛋白连接蛋白40（CX40）在连接黄斑densa和传入的动脉的肾小管上皮的外部肌外膜中高度表达。 CX40参与并非肾素表达增加，表达肾素细胞的定位在CX40缺陷小鼠中异常。此外，发现在两种不同遗传背景的CX40缺陷小鼠中，TGF反应性的大小降低，并以响应速度减慢，这表明间隙连接性通信可能有助于信号传递。 5。黄斑densa上游的管状片段中的流体重吸收是JGA依赖性调节的一部分，因为它确定了输送到传感位点的NaCl载荷。我们先前已经表明，通过提供跨膜进入和水的出口途径，需要高吸收率的近端小管中的水通道蛋白1。但是，尚不清楚近端小管中的水是否也可以通过副细胞途径穿过上皮。 Claudin2（CLDN2）作为在肾脏近端小管中高度表达的紧密连接蛋白的鉴定，以及证据表明CLDN2增加培养细胞中水的渗透性的证据促使我们促使我们比较了野生型和CLDN2缺乏型小鼠中近端流体的吸收。我们发现，CLDN2依赖性的液体的重吸收占总重吸收的27.5％，这表明近端流体转运的副细胞旁途径。 CLDN2缺陷型小鼠的液体重吸收减少与GFR降低有关，这可能是由于向TGF传感器递送盐的结果。我们目前正在生成具有含水通道蛋白1和Claudin 2的删除的小鼠，以检查膜或紧密连接蛋白介导的液体吸收成分是否是独立且添加剂的。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Annexin A1 modulates macula densa function by inhibiting cyclooxygenase 2.

膜联蛋白 A1 通过抑制环氧合酶 2 来调节致密斑功能。

DOI：
10.1152/ajprenal.00704.2011
发表时间：
2012
期刊：
American journal of physiology. Renal physiology
影响因子：
0
作者：
Seidel,S;Neymeyer,H;Kahl,T;Roschel,T;Mutig,K;Flower,R;Schnermann,J;Bachmann,S;Paliege,A
通讯作者：
Paliege,A

Renin expression in large renal vessels during fetal development depends on functional beta1/beta2-adrenergic receptors.

胎儿发育期间大肾血管中肾素的表达取决于功能性 β1/β2 肾上腺素能受体。