Symptoms and hospitalizations for heart failure (HF) are primarily driven by congestion, making loop diuretics a cornerstone therapy in HF. This is problematic since loop diuretic resistance (DR) is common and a driver of persistent congestion and the poor outcomes that follow. We have recently confirmed that: 1) The dominant driver of DR in human HF is at the renal tubular level, rather than poor diuretic delivery. 2) Proximal tubular sodium reabsorption is not a substantial contributor, rather 3) reduced response at the site of action in the loop of Henle and compensatory distal tubular sodium reabsorption drive DR. 4) Resistance at the loop of Henle appears to be addressable with diuretic doses traditionally considered above the ceiling dose. Despite progress in defining the general locations for DR, the culprit transporters and thus specific druggable targets remain undefined. There is consensus on the existence of three stoichiometrically relevant distal sodium (Na) transport pathways. The central components to these three pathways are the sodium chloride cotransporter (NCC), the epithelial sodium channel (ENaC), and the chloride bicarbonate exchanger, pendrin. Importantly, these targets can be manipulated in humans with FDA approved drugs; NCC can be selectively inhibited by bendroflumethiazide (a thiazide with minimal carbonic anhydrase inhibition), ENaC by amiloride, and pendrin downregulated by NH4Cl loading. We have also learned that sodium reabsorption in the loop of Henle is dynamic with substantial regulation and plasticity of NKCC2. Importantly, NKCC2 splice variants have been identified that have dramatically different ion affinities, transport capacity, and diuretic sensitivities. The primary goal of this proposal is to translate the above knowledge into therapeutically actionable approaches to human DR. To accomplish this, we will conduct 3 mechanistically focused clinical trials using pharmacologic manipulation of different transport pathways, endogenous lithium clearance to understand regional nephron sodium handling, and urinary extracellular vesicles to investigate differences in tubular solute transporter levels and splice variants. Specifically, Aim 1 will investigate the mechanism underlying the substantial shift in the loop diuretic dose response curve to the right in human HF. Here we will serially titrate the highly selective NKCC2 antagonist bumetanide to 10mg (400mg furosemide equivalents) in stable DR and diuretic responsive HF patients. In Aim 2 we seek to understand the effect of acute antagonism of amiloride sensitive, thiazide sensitive, or combined amiloride & thiazide sensitive transport pathways on loop diuretic response in stable DR HF patients. We will accomplish this by administration of the combinations of placebo, amiloride, and/or bendroflumethiazide, in conjunction with bumetanide, to stable DR HF patients. In Aim 3 we will determine if NH4CL loading, known to downregulate pendrin, can reduce non-amilo...