Lipid Rafts: Mechanosensors of the distal nephron

高血压（HTN）影响美国数百万成年人，并导致终末器官损伤。本研究 旨在测试胆固醇（chol）流出途径的失调是否影响钠（Na）依赖性 全身血压（BP）升高，两者都是死亡的危险因素。 chol流出途径由两条途径组成，（1）促进chol转运或（2）激活chol转运。 由ATP结合盒（ABC）转运蛋白、ABC转运蛋白A1（ABCA 1）和 ABC转运蛋白G1（ABCG 1）。ABCA 1多态性，抑制胆固醇流出，与HTN相关， ABCA 1的非功能性突变导致Tangiers病和早期动脉粥样硬化。 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，增加钠排泄;作为必然结果，靶向删除流量介导的自分泌-旁分泌途径 增强肾Na亲和力和HTN。肾前列腺素E2（PGE 2）敲除（KO）导致Na敏感 HTN。此外，肾小管血流的增加诱导环氧化酶-2（COX 2）活性/蛋白和集合管 (CD)PGE 2释放以抑制上皮Na通道（ENaC）。流动对COX 2和PGE 2的影响是静音的， 在从喂食1%胆固醇与不喂食胆固醇饮食的小鼠中分离的CD中。与对照组相比， 对照CD中ABCA 1消融增加CD胆固醇，刺激ENaC，减少尿ATP并升高BP。 ABCA 1消融也易导致炎症性肾脏微环境。游离LR增加 胆固醇增强与肾小管Na转运相关的配体依赖性Toll样受体（TLR）4信号传导。 升高的LR游离胆固醇将TLR 4募集至LR，增加LR TLR 4密度，以增强信号传导。经上皮 Na转运刺激Na/K-ATP酶介导的ATP需求，驱动线粒体氧化呼吸， 氧化应激、脂质过氧化和铁缺乏症。TLR 4被应激诱导的配体刺激，包括脂质 过氧化物导致炎性和嗜钠表型。因此，我们假设， ABCA 1缺陷小管中的PM/LR胆固醇含量将诱导Na亲和力和炎症， 流量介导的利钠途径，引发的TLR 4反应，和转运依赖性氧化 呼吸和胁迫形成Na敏感表型。这将在具体目标中得到检验： SA 1.为了评估肾小管ABCA 1表达的缺失或缺失是否有助于肾小管上皮细胞的凋亡， 肾嗜钠性和HTN的发展 SA1a。测试肾小管ABCA 1消融是否足够或饮食胆固醇是否需要诱导钠敏感性。 SA1b.通过将ABCA 1 fl/fl（ABCA 1 FF）小鼠与 载脂蛋白E缺陷小鼠 SA 2.测试chol是否影响PM/LR功能以抑制流量介导的Na排泄途径， 增强TLR 4炎症信号传导以促进抗钠尿和HTN SA2a。测试胆固醇掺入LRs是否直接增强Na转运并抑制诱导的FSS 利用分子、生理学和电生理学方法研究利钠因子对钠吸收的影响。 SA2.b确定改变PM chol，特别是LR chol是否影响TLR 4密度和活化， 以及炎症标志物和跨上皮钠转运。 SA2.c测试药理学PM胆固醇消耗是否逆转钠敏感性和炎症。 SA 3. ABCA 1缺陷小鼠的Na转运速率增加，增加氧化呼吸和应激 基于单核RNAseq（snRNAseq）数据，激活铁离子途径。 SA 3a。确定更高的Na/K-ATP酶活性是否驱动更大的线粒体呼吸， ABCA 1缺乏的肾脏中的氧化应激。 SA 3b.表征线粒体呼吸和应激增加是否会增加肾小管上皮细胞的铁凋亡。

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.