Lipid Rafts: Mechanosensors of the distal nephron

高血压(HTN)在美国影响数以百万计的成年人，并导致最终的器官损伤。这项研究是 旨在测试胆固醇(CHOL)外流途径的失调是否会影响钠(Na)依赖 全身血压(BP)升高，两者都是死亡的风险因素。 Chol外流途径由两条途径组成，(1)促进Chol转运或(2)主动转运 三磷酸腺苷结合盒(ABC)转运体、ABC转运体A1(ABCA1)和 ABC转运蛋白G1(Abcg1)。抑制胆固醇外流的ABCA1基因多态与HTN有关，而 ABCA1的非功能突变会导致早期动脉粥样硬化的丹吉尔病。 高钠饮食增加肾小管流量，刺激旁分泌途径抑制钠吸收， 因此，增加钠的排泄；作为推论，有针对性地删除流动介导的自分泌-旁分泌途径 增强肾脏钠亲和力和HTN。肾前列腺素E2(PGE2)基因敲除(KO)引起钠敏感 HTN.此外，肾小管流量增加可诱导环氧合酶-2(COX2)活性/蛋白和集合管 (Cd)释放PGE2以抑制上皮钠通道(ENaC)。流动对COX2和PGE2的影响减弱 从喂食1%胆固醇饮食的小鼠与不喂食胆固醇饮食的小鼠分离的CD中。喂饲胆固醇的小鼠的胆固醇含量是 控制。ABCA1消融CD，增加Cd胆固醇，刺激ENaC，降低尿三磷酸腺苷，升高血压。 ABCA1消融术也易导致炎症的肾脏微环境。增加LR空闲量 Chol增强配体依赖的Toll样受体(TLR)4信号，该信号与肾小管钠转运有关。 升高的LR游离CHOL将TLR4招募到LR，增加LR TLR4的密度，以增强信号转导。跨上皮 Na转运刺激Na/K-ATPase介导的ATP需求驱动线粒体氧化呼吸， 氧化应激、脂质过氧化和铁性下垂。TLR4受包括脂质在内的应激诱导配体的刺激 过氧化物质导致炎症和嗜钠表型。因此，我们假设多余的肾小管 ABCA1缺陷性肾小管PM/LR胆固醇含量降低会引起钠离子亲和性和炎症反应 流动介导的利钠途径、启动的TLR4反应和运输依赖的氧化 呼吸作用和胁迫形成钠敏感的表型。这将在特定目标(SA)中进行测试： SA1.为了评估肾小管ABCA1表达的缺失或缺失是否有助于 肾脏钠亲和力与HTN的发展 SA1a.测试是否管状ABCA1消融是足够的，还是饮食中的胆固醇是诱导钠敏感性的必要因素。 SA1b.用ABCA1fl/fl(ABCA1Ff)小鼠杂交来确定血清低密度脂蛋白水平是否改变钠亲和力 载脂蛋白E(ApoE)缺陷小鼠 SA2.检测CHOL是否影响PM/LR功能以抑制流介导的钠排泄途径和 增强TLR4炎症信号促进抗钠尿和HTN SA2a。检测胆醇掺入LRS是否直接促进钠转运并抑制FSS诱导的 利用分子、生理学和电生理学方法增加钠吸收的利钠因子。 SA2.b确定改变PM CHOL，特别是LR CHOL是否影响TLR4密度和激活， 因此，炎症标记物和跨上皮钠的运输。 SA2.c测试药物性PM胆固醇耗竭是否逆转钠敏感性和炎症。 SA3.ABCA1缺陷小鼠较高的钠转运速率增加了氧化呼吸和应激 这激活了基于单核RNAseq(SnRNAseq)数据的铁电性途径。 Sa3a.确定更高的Na/K-ATPase活性是否推动了更大的线粒体呼吸 ABCA1缺陷肾脏的氧化应激。 SA3b.表征线粒体呼吸增加和应激是否会增加肾小管上皮细胞的铁性下垂。

Hypertension (HTN) affects millions of adults in the U.S and leads to end organ damage. This study is designed to test whether dysregulation of the cholesterol (chol) efflux pathway affects sodium (Na) dependent rise in systemic blood pressure (BP), both of which are risk factors for mortality. The chol efflux pathway is comprised of two pathways, (1) facilitated chol transport or (2) the active transport pathway regulated by ATP-binding cassette (ABC) transporters, ABC transporter A1 (ABCA1) and ABC transporter G1 (ABCG1). ABCA1 polymorphisms, that repress chol efflux, are associated with HTN while non-functional mutations of ABCA1 cause Tangiers disease with early atherosclerosis. High Na diets increase tubular flow that stimulate paracrine pathways to suppress Na absorption and, thus, enhance Na excretion; as a corollary, targeted deletion of flow-mediated autocrine-paracrine pathways enhance renal Na avidity and HTN. Renal prostaglandin E2 (PGE2) knock out (KO) causes Na sensitive HTN. Moreover, increases in tubular flow induce cyclooxygenase-2 (COX2) activity/protein and collecting duct (CD) PGE2 release to inhibit epithelial Na channel (ENaC). The effects of flow on COX2 and PGE2 are muted in CDs isolated from mice fed a 1% chol vs. no chol diet. The chol content from chol fed mice doubles versus controls. ABCA1 ablation in CDs, increases CD chol, stimulates ENaC, reduces urinary ATP and raises BP. ABCA1 ablation also predisposes to an inflammatory renal microenvironment. Increases of LR free chol enhance ligand-dependent toll-like receptor (TLR) 4 signaling which is linked to tubular Na transport. Elevated LR free chol recruit TLR4 to LRs, increasing LR TLR4 density, to augment signaling. Transepithelial Na transport stimulates Na/K-ATPase mediated ATP demand driving mitochondrial oxidative respiration, oxidative stress, lipid peroxidation and ferroptosis. TLR4 is stimulated by stress induced ligands including lipid peroxides leading to an inflammatory and Na avid phenotype. Thus, we hypothesize that excess tubular PM/LR chol content in ABCA1 deficient tubules will induce Na avidity and inflammation due to muted flow mediated natriuretic pathways, primed TLR4 responses, and transport dependent oxidative respiration and stress to form the Na sensitive phenotype. This will be tested in the specific aims (SAs): SA1. To evaluate whether depletion or deletion of tubular ABCA1 expression contributes to the development of renal Na avidity and HTN SA1a. Test whether tubular ABCA1 ablation is sufficient or dietary chol is necessary to induce Na sensitivity. SA1b. Determine whether serum LDL level modifies the Na avidity by crossing ABCA1fl/fl (ABCA1 FF) mice into the apolipoprotein E (APOE) deficient mouse SA2. Test whether chol affects PM/LR function to repress flow mediated Na excretory pathways and augment TLR4 inflammatory signaling to promote anti-natriuresis and HTN SA2a. Test whether chol incorporation into LRs directly enhances Na transport and represses FSS induced natriuretic factors to augment Na absorption using molecular, physiologic and electrophysiologic methods. SA2.b Determine whether altering the PM chol, and specifically, LR chol effects TLR4 density and activation, and, hence, inflammatory markers and transepithelial Na transport. SA2.c Test whether pharmacologic PM chol depletion reverses Na sensitivity and inflammation. SA3. Greater rates of Na transport in ABCA1 deficient mice increase oxidative respiration and stress that activates the ferropotic pathway, based on single nuclei RNAseq (snRNAseq) data. SA3a. Determine whether greater Na/K-ATPase activity is driving greater mitochondrial respiration and oxidative stress in ABCA1 deficient kidneys. SA3b. Characterize if increased mitochondrial respiration and stress augments ferroptosis in tubular epithelia.