PROJECT SUMMARY The Na+/K+/2Cl- co-transporter (NKCC2) is the main sodium transporter in the medullary thick ascending limb (mTAL), and enhanced activity of NKCC2 has been linked to hypertension in animal models and humans. We recently found mutant ACTN4—a cytoskeletal protein previously known to contribute to human podocyte dysfunction and glomerular disease—leads to salt sensitive hypertension in a novel rat model. ACTN4 is expressed in mTAL, and we found in our new adaptation of organ-on-chip technology, mTAL-on-chips, that mutant ACTN4 contributes to increased sodium uptake within mTAL cells via enhancing NKCC2 activity. Our long-term goal is to define the role of ACTN4 in regulating sodium homeostasis and blood pressure salt sensitivity. The overall objective of this application is to determine the degree to which disease-causing mutant ACTN4 influences sodium handling in mTAL. Our central hypothesis is that mutant ACTN4 alters the F-actin cytoskeletal network, which in turn disrupts normal trafficking of NKCC2 within mTAL cells. This disruption of trafficking leads to accumulation of NKCC2 on the apical membrane of mTAL cells, elevated sodium reabsorption in mTAL, and ultimately salt-sensitive hypertension. Aim 1 will determine the degree to which mutant ACTN4 increases salt-sensitivity in mTAL cells. We will use mTAL-on-chips seeded with wild type (WT) mTAL cells and mutant ACTN4 mTAL cells. We will measure sodium reabsorption of mTAL cells in response to varying levels of stress, including increasing sodium concentration, shear stress, and stretch. Aim 2 will elucidate the mechanism by which mutant ACTN4 disrupts NKCC2 trafficking. We will measure differences between WT mTAL and mutant ACTN4 mTAL cells in (1) the cell surface NKCC2 levels and (2) NKCC2 trafficking (endocytosis and exocytosis). We will further measure whether these differences are amplified in response to varying levels of stress. Aim 3 will ascertain whether in vivo salt-sensitive hypertension associated with mutant ACTN4 is mediated by NKCC2. We will examine the effect of mutant ACTN4 on pressure natriuresis. We will also assess the effect of inhibiting NKCC2 with furosemide on acute natriuresis and blood pressure salt-sensitivity. The research proposed in this application is innovative in that it integrates the novel mTAL-on-chip technology with advanced imaging and whole animal physiologic studies to comprehensively delineate the mechanism by which mutant ACTN4 leads to salt-sensitive hypertension. This research will provide strong evidence of a novel and essential mechanism of sodium and blood pressure regulation performed by the cytoskeletal protein ACTN4.