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 Determin...