Urea transporters (UTs) are expressed in kidney tubules and microvessels, and in extrarenal tissues. An intrarenal urea recycling process through urea transporters (UTs) is believed to be important in the urinary concentrating mechanism and thus water conservation. We recently generated and analyzed UT-B deficient mice, the first animal model of UT deficiency. Our data provided evidence that UT-B-dependent countercurrent exchange of urea in the renal medulla contributes to approximately one-third of the kidney's total capacity to concentrate urine, but contributes even more greatly to the ability of the kidney to concentrate urea itself. The purpose of this proposal is to generate transgenic mice that lack other UTs, and to characterize their role in the kidney. In Aim 1, we propose to generate: UT-A2 knockout, UT-A2/UT-B double knockout and UT-A1/A3/A4 triple knockout mice by various targeting strategies. The generation of these mice is technically feasible and appropriate to address specific questions in renal physiology: UT-A2 and UT-A2/UT-B knockout mice to study urea recycling and urea reintroduction into descending limb of Henle's loops and vasa recta, and UT-A1/A3/A4 triple knockout mice to study urea delivery to the tip of the papilla. In Aim 2, renal function in wild type and knockout mice will be studied systematically by classical urine/plasma collection methods using metabolic cages. The urinary concentrating mechanism will be analyzed under normal and stressed conditions (water deprivation, water-loading, high/low protein diet). These experiments will permit the definitive testing of hypotheses regarding the role of intrarenal urea recycling in the urine concentrating mechanism (roles of urea efflux from collecting duct and urea influx into vasa recta and descending limb of Henle's loops), and provide the first quantitative data on the role of specific UTs. Together the data should provide novel and definitive data regarding the role of specific UTs in mammalian renal physiology and new insights into pathological conditions such as chronic renal failure and hypertension. Also, the new information may provide a rational basis for discovery of UT inhibitors as unique diuretic agents.