The generation of a concentrated urine by the kidney involves a countercurrent multiplication mechanism, which is facilitated by urea transporter (UT)-A-type urea transporters in tubule epithelial cells, and a countercurrent exchange mechanism, which is facilitated by UT-B in microvascular (vasa recta) endothelia. Loss of UT function is predicted to disrupt urinary concentrating ability. We propose UTs as novel targets for the development of a new type of diuretic, which we call 'urearetic', with a novel mechanism of action and a unique clinical indication profile. The primary goal of this proposal is to deliver drug-like, validated UT inhibitors for clinical development. Additional deliverables include the generation of potent UT-selective inhibitors as research tools, and, using these tools, to generate new data on renal UT physiology in rodent models by chemical knockout, recognizing its advantages over gene knockout.
In Aim 1, a novel high-throughput screen will be used to identify UT inhibitors with different UT isoform selectivity profiles.
This aim follows from extensive preliminary data on assay development and identification of UT-B and UT-A1 inhibitors. Screening against each of the major renal UT isoforms, UT-A1, UT-A2 and UT-A3 to identify active compounds for study structure-activity relationships and selectivity profiles will e conducted.
In Aim 2, UT inhibition mechanisms and pharmacology of compounds identified in Aim 1 will be determined in order to establish a prioritized list of compounds for animal testing. Target compound properties include high UT inhibition potency (low nM IC50) and good pharmacological profile. Studies will include: (a) using cell culture models - inhibition reversibility, kinetics, sidedness and urea competition; (b) by computational chemistry - the molecular basis of inhibition potency and selectivity; and (c) using rats - pharmacokinetics, renal/urine accumulation and toxicity.
In Aim 3, rodent models and UT inhibitors will be used to characterize the role of UTs in urinary concentrating function and to obtain proof-of-concept for UT inhibitor therapy of edema. Target effects of UT inhibitors in vivo include increasing urine output and reducing urinary concentrating ability, and reducing edema in clinically relevant states of fluid accumulation. Studies in rats will include measurements of compound effects on urine output, osmolality and urea concentration, and serum urea concentration, during normal hydration and with dehydration DDAVP. Compound(s) will also be tested in a rat model of edema in congestive heart failure. The outcomes of this proposal will include drug-like, validated UT inhibitors for use as research tools and for clinical development, and new information on the role of UTs in the urinary concentrating mechanism.
Urea transporters play an important role in the generation of concentrated urine by the kidney. The goal of this proposal is to identify and optimize drug-like urea transport inhibitors, and to obtain proof-of-concept in animal models for their application as a new type of diuretic, with a novel mechanism of action, to treat states of fluid overload such as in congestive heart failure.
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