The epithelial Na+ channel is the rate limiting step for Na+ uptake and tubular Na+ reabsorption by the distal nephron of the kidney and is a major site for hormonal regulation of extracellular salt and water, thereby playing a critical role in a fundamental homeostatic function of the mammalian kidney. The underlying mechanisms of aldosterone regulation of channel activity in the kidney are not well understood. The major goal of the proposed studies is to define by immunocytochemical methods the tubular and cellular distribution of Na+ channel protein in the structurally heterogeneous rat kidney, and to evaluate mechanisms of regulation of the channel by aldosterone. These studies will use antibodies to submit-specific peptide sequences based on recent cloning of Na channel subunits in the Rossier laboratory. The specific aims of the research will be 1) to generate new antibodies to several of the subunits using a method in which multiple copies of subunit-specific sequence are synthesized on a polylysine core to provide the source of antigen: 2) to examine the immunocytochemical distribution of channel subunits by conventional and confocal fluorescence microscopy in normal rat kidney; and 3) to characterize alteration in the expression and/or distribution of channel protein in kidneys from animals maintained on a low salt diet to elevate serum aldosterone levels. The results will be used to distinguish between proposed regulatory mechanisms, including de novo synthesis and recruitment of channels (or subunits) from a cytosolic store.