The specific aims of this FIRCA proposal are: 1) To isolate pure endocytic vesicles from different kidney regions (cortex, inner stripe of the outer medulla, papilla). Vesicles purified by traditional methods are contaminated with other membranes, and we plan to develop and use affinity- purification techniques with specific antibodies to improve vesicle purity while maintaining a high yield; 2) To characterize enzyme and transport activities of the resulting endosomal preparations, including the H+-ATPase (H-pump), Na+/H+-exchange, Ca2+ /H+-exchange, Ca2+=ATPase (Ca-pump), Cl- channel, and different types of water channels; 3) To generate immune sera by immunizing rabbits with preparations of highly purified endocytic vesicles; 4) To purify particular antibodies by immunoadsorption techniques from immune sera. A battery of antibodies, thus obtained, will be used in immunocytochemical studies on renal tissue sections in order to specify one or more antibodies that specifically label endocytic vesicles in defined populations of renal cells. These antibodies will be used as tools for the further affinity purification of vesicle populations as well as for studies on vesicle transport pathways that are part of the parent grant. The results will allow us to describe in more detail the molecular and functional heterogeneity of endosomal vesicles from various tubules in the mammalian kidney and to study their behaviour in a variety of conditions with disturbances in salt, water, and protein reabsorption.