Lipid Rafts: Mechanosensors of the distal nephron

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 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.