Lipid Rafts: Mechanosensors of the distal nephron

 描述(由申请人提供)： 机械力，特别是流体切应力(FSS)，在调节血管反应性的内皮细胞和影响跨上皮阳离子转运的肾上皮细胞中发挥关键作用，这些跨上皮阳离子转运共同决定了血压(BP)。肾小管阳离子转运的机械调节是肾脏钠(Na)稳态的重要调节因素，提示肾上皮细胞存在机械感受器。富含胆固醇(Chol)的脂筏(LRs)是位于质膜(PM)上的信号平台，是诱导血管内皮细胞旁分泌介导的血管扩张(即一氧化氮合成)的机械感受器；然而，人们对它们在血管内皮细胞中的感觉功能知之甚少。 肾上皮细胞。Chol维持LRs的完整性，而对LR Chol含量的影响影响FSS介导的信号转导。调节细胞内丝裂原活化蛋白激酶(MAPK)和细胞内钙离子浓度([Ca+]i)的FSS敏感信号通路(1)分别定位于小窝蛋白-1(Cav-1)和FLOT-2(FLOT-2)，在集合管(CD)细胞中表达LRs；(2)分别控制COX-2表达和PGE2释放的变化。此外，CHOL整合到表达LRs的E-前列腺素(EP)受体可以改变调节跨上皮钠吸收的信号。在初步研究中，在胆固醇喂养的小鼠中发现了一种抗利尿剂、高血压的表型，经肠外生理盐水扩张，这与显微解剖的皮质CD(CCDs)中流动介导的COX-2活性和PGE2释放受到抑制有关。因此，最终完整的上皮细胞对肾小管流速的反应取决于CHOL校准、FSS介导的PGE2释放和调节Na转运的EP受体刺激。我们推测，高胆固醇血症改变了肾CD受体的CHOL含量，从而调节Flow/FSS诱导的COX-2表达和PGE2的释放，而PGE2通过自分泌/旁分泌的EP受体信号影响远端肾单位的钠转运。这一假设将在以下特定目标(SA)中进行检验：SA1。检测富含胆固醇的受体是否是FSS介导的刺激多囊蛋白-2(PC-2)和MAPK激活的机械传感平台。SA1.a评估PC-2和MAPK(p38，ERK)在CD细胞和显微解剖的CD细胞中特定LRs群体中的定位。PC-2是一种机械敏感通道，负责钙离子进入CD细胞的顶端。SA1.b确定FSS是否导致PC-2和MAPK移位出各自的富含胆固醇的受体，以允许最大限度地激活PC-2和MAPK磷酸化。SA1.c测试LR胆固醇含量是否影响静息定位和FSS诱导的PC-2和MAPK移位出LR。SA2.测试短期摄入胆固醇是否会导致胆碱能团结合，从而改变血流介导的COX刺激、PGE2释放和钠排泄。SA2.a评估饮食中胆固醇的摄入量是否包含在肾脏CCDSA2.b中，确定整合到肾脏CCDs中的胆固醇是否改变了PGE2的净生成能力、流动介导的PGE2释放、钠排泄，进而改变了血压。SA3.测试短期摄入胆固醇是否抑制血流介导的PGE2释放和改变EP受体依赖的信号转导，以增强微灌流CCDS SA3.a的跨上皮钠转运。a评估CCDs中胆固醇的掺入是否抑制血流介导的PGE2释放以促进钠的吸收。SA3.b测试CCDs中CHOL掺入是否改变EP受体信号以增强Na的吸收。

 DESCRIPTION (provided by applicant): Mechanical forces and, in particular, fluid shear stress (FSS), play critical roles in endothelia t regulate vascular reactivity and in renal epithelia to affect transepithelial cation transport that collectively, determine blood pressue (BP). Mechano-regulation of tubular cation transport is an important regulator of renal sodium (Na) homeostasis and infers the existence of mechano-sensors in renal epithelia. Cholesterol (chol)-rich lipid rafts (LRs), signaling platforms located n the plasma membrane (PM), are mechano-sensors that induce paracrine-mediated vasodilation (ie nitric oxide synthesis) in endothelia; however, little is known about their sensory function in renal epithelia. Chol maintains the integrity of LRs, and effects on LR chol content impacts FSS-mediated signaling. The FSS-sensitive signaling pathways which regulate mitogen activated protein kinase (MAPK) and intracellular Ca2+ concentration ([Ca2+]i) in cells (1) are localized to caveolin-1 (cav-1) and flotillin-2 (flot-2), respectively, expressing LRs in collecting duct (CD) cells, and (2) control changes in COX-2 expression and PGE2 release, respectively. Additionally, chol integration into E-prostanoid (EP) receptor expressing LRs can alter signaling that regulates transepithelial Na absorption. In PRELIMINARY STUDIES an anti-diuretic, hypertensive phenotype was uncovered in chol fed mice, volume expanded by parenteral saline, that was associated with suppressed flow-mediated COX-2 activity and PGE2 release in microdissected cortical CDs (CCDs). Thus, the final integrated epithelial response to tubular flow rate is determined by chol calibrated, FSS-mediated PGE2 release and EP receptor stimulation which regulate Na transport. We hypothesize that hypercholesterolemia alters the chol content in LRs of the renal CD to regulate flow/FSS-induced COX-2 expression and PGE2 release which, via autocrine/paracrine EP- receptor based signaling, influences Na transport in the distal nephron. This hypothesis will be tested in the following specific aims (SAs): SA1. Test whether chol-rich LRs are mechanosensory platforms for FSS-mediated stimulation of polycystin-2 (PC-2) and MAPK activation. SA1.a Evaluate the localization of PC-2, a mechano-sensitive channel responsible for apical entry of Ca2+ into CD cells, and MAPK (p38, ERK) within specific populations of LRs in CD cells and microdissected CCDs. SA1.b Determine whether FSS leads to translocation of PC-2 and MAPK out of their respective chol-rich LRs to allow for maximal PC-2 activation and MAPK phosphorylation. SA1.c Test whether LR chol content affects resting localization and FSS-induced translocation of PC-2 and MAPK out of LRs. SA2. Test whether short-term chol ingestion leads to incorporation of chol into the CCD that alters flow-mediated COX stimulation, PGE2 release and Na excretion. SA2.a Evaluate whether dietary chol consumption incorporates into the renal CCD SA2.b Determine whether chol integration into renal CCDs modifies net PGE2 generating capacity, flow- mediated PGE2 release, Na excretion and, in turn, BP. SA3. Test whether short-term chol ingestion inhibits flow-mediated PGE2 release and alters EP receptor dependent signaling, to enhance transepithelial Na transport in microperfused CCDs SA3.a Evaluate whether chol incorporation in CCDs suppresses flow-mediated PGE2 release to augment Na absorption. SA3.b Test whether chol incorporation in CCDs modifies EP receptor signaling to enhance Na absorption.