Lipid Rafts: Mechanosensors of the distal nephron

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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.